Chiral peptide nucleic acids

ABSTRACT

Chiral peptide nucleic acids are provided which hybridise strongly with complementary nucleic acids and have potential as antigene and antisense agents and as tools in molecular biology. Compounds with cis-stereochemistry and based on proline and a spacer amino acid have structures (II), (III), where n is 1 or 2-200, B is a protected or unprotected base, R is H or alkyl, aralkyl or heteroaryl and may be substituted, X may be OH, and Y may be H.

INTRODUCTION

[0001] This invention describes the synthesis and properties of a novelclass of chiral peptide nucleic acids (cPNAs) which hybridise stronglywith complementary nucleic acids. As such they have potential asantigene and antisense agents and as tools in molecular biology.

[0002] Oligonucleotides are potentially useful for the regulation ofgenetic expression by binding with DNA or mRNA¹. However, naturaloligonucleotides are degraded by nucleases, consequently there isconsiderable interest in synthetic oligonucleotide analogues which arestable under physiological conditions. Recently, there has been interestin oligonucleotide analogues in which the sugar-phosphate backbone isreplaced by a peptide chain² after the success of the so-called PeptideNucleic Acids (PNA)³, but more correctly referred to as PolyamideNucleic Acids⁴.

[0003] The sugar phosphate backbone of a nucleic acid consists of arepeating unit of six atoms, configurationally and conformationallyconstrained by the D-ribose or 2′-deoxy-D-ribose ring. If this could bereplaced by a dipeptide unit the new backbone would be amenable topreparation by solid phase peptide synthesis. Molecular modelling bycomputer graphics suggested that a peptide chain consisting of analternate sequence of a “nucteo-amino acid” derived from proline and a“spacer amino acid”, which could be any amino acid, should be a suitablestructural analogue of the ribose phosphate backbone of nucleic acids asshown.

SUMMARY OF INVENTION

[0004] This invention provides compounds of formula (I)

[0005] where n is 1 or 2-200

[0006] B is a protected or unprotected heterocyclic base capable ofWatson-Crick or of Hoogsteen pairing,

[0007] R is H, C1-C12 alkyl, C6-C12 aralkyl or C6-C12 heteroaryl whichmay carry one or more substituents preferably selected from hydroxyl,carboxyl, amine, amide, thiol, thioether or phenol,

[0008] X is OH or OR′ where R′ is a protecting group or an activatinggroup or a lipophilic group or an amino acid or amino amide ornucleoside,

[0009] Y is H or a protecting group or a lipophilic group or an aminoacyl group or nucleoside.

[0010] When n is 1, these compounds are peptide nucleotide analogues.When n is 2 to about 30 these compounds are peptide oligonucleotides,which are synthesised as described below and can be hybridised toordinary oligo or polynucleotides. Typically the two strands arehybridised to one another in a 1:1 molar ratio by base-specificWatson-Crick base pairing.

[0011] B is a base capable of Watson-Crick or of Hoogsteen pairing. Thismay be a naturally occurring nucleobase selected from A,C,G,T and U; ora base analogue that may be base specific or degenerate, e.g. by havingthe ability to base pair with both pyrimidines (T/C) or both purines(A/G) or universal, by forming base pairs with each of the natural baseswithout discrimination. Many such base analogues are known, e.g.hypoxanthene, 3-nitropyrrole, 5-nitroindole, and those cited by Lin andBrown⁵ and all are envisaged for use in the present invention.

[0012] The compounds of formula (I) contain proline of undefinedstereochemistry. Although compounds with the trans-stereochemistry mayhave interesting properties, compounds with the cis-stereochemistry arepreferred either with the D-configuration as shown in (II) or theL-configuration shown in structure (III). In these compounds bothstereoisomers of the “spacer amino acid” NHCHRCO are envisaged.

[0013] Provided that it does not sterically hinder chain extension ofhybridisation, the group R could have diverse structures. The group,however, can be chosen to confer desired hydrophobic, hydrophilic and/orelectrostatic properties on the molecule. When the group R is other thanH it generates a chiral centre and the two stereoisomers may allow fordiscrimination in the hybridisation of DNA and RNA. When the amino acid(—NH—CHR—CO—) is a naturally occurring amino acid, then the amino acidshould be readily and cheaply available as a building block forcompounds of this invention. Any of the natural or unnatural α-aminoacids could be used e.g. glycine or L- or D-serine or lysine. The natureof X can be varied from the negatively charged carboxylate ion (X═O⁻) tothe incorporation of a positively charged lysine residue. Examples ofthe latter are provided in the experimental section and can be used toprevent aggregation and to assist hybridisation to the negativelycharged oligonucleotides. Y will most commonly be H but could be anygroup which might be useful to improve the physical or biologicalproperties of the material.

[0014] Any one of B, R, X and Y may include a signal moiety, which maybe for example a radioisotope, an isotope detectable by massspectrometry or NMR, a hapten, a fluorescent group or a component of achemiluminescent or fluorescent or chromogenic enzyme system. The signalmoiety may be joined to the peptide nucleotide analogue either directlyor through a linker chain of up to 30 atoms as well known in the field.

[0015] In another aspect the invention provides a method of making thepeptide nucleotide analogue of formula (I), comprising the steps of:

[0016] a) reacting an N-protected C-protected 4-hydroxy proline with abase selected from N₃-protected thymine, N₆-protected adenine,N₄-protected cytosine, N₂—O₆-protected guanine and N.-protected uracil.

[0017] b) deprotecting the proline amino group of the product of a),

[0018] c) reacting the product of b) with an N-protected amino acid,

[0019] d) optionally removing protecting groups from the product of c).

[0020] In another aspect the invention provides a method of converting apeptide nucleotide analogue of formula (I) in which n is 1 into apeptide oligonucleotide of formula (I) in which n is 2-200, comprisingthe steps of:

[0021] i) providing a support carrying primary amine groups,

[0022] ii) coupling an N-protected peptide nucleotide analogue offormula (I) to the support,

[0023] iii) removing the N-terminal protecting group,

[0024] iv) coupling an N-protected nucleotide analogue of formula (I) tothe thus-derivatised support,

[0025] v) repeating steps iii) and iv) one or more times, and

[0026] vi) optionally removing the resulting peptide oligonucleotidefrom the support.

[0027] The invention also provides a compound of formula (IV)

[0028] where R² is H or a protecting group,

[0029] R³ is H or a protecting group compatible with R², and

[0030] B is a protected or unprotected heterocyclic base.

[0031] The invention also provides a compound of formula (V)

[0032] where R² is diphenylmethyl, and

[0033] R³ is t-butoxycarbonyl.

DETAILED DESCRIPTION

[0034] The (2R,4R) (“cis-D”)-proline was chosen since this is analogousto the stereochemistry of deoxyribonucleotides. The lack of negativecharge on the peptide backbone would be expected to lead to a higheraffinity for complementary oligonucleotide sequences in nucleic acids.Moreover these novel peptide nucleic acids can also be modified easilyby using different “spacer amino acids” to affect physical andbiological properties such as solubility, cell permeability, etc. inorder to achieve higher therapeutic activity. Such peptide nucleic acidsshould be stable to proteases since they contain substituted D-prolineresidues at alternate sites. Since coupling to secondary amino acids canbe slow and inefficient, it was decided to use dipeptide building blocksin which the amino-acyl-proline bond is formed in solution as indipeptide (1). In the alternative arrangement, i.e. prolyl-amino acid,there is likely to be a serious problem of racemisation during thecoupling if the amino acid is chiral, whereas such racemisation is notexpected when the C-terminus of the activated fragment is prolinebecause N-acylprolines can not racemise by the oxazolone mechanism⁶.

[0035] Because of the mild conditions used for the deprotection of theN-Fmoc group, the Fmoc/O'Bu strategy in solid phase peptide synthesis isfavoured over the classical Boc/OBzl strategy⁷. Furthermore, mostmachine synthesisers capable of handling small scale synthesis (50 μmolor less) can accommodate only the Fmoc/O'Bu strategy. For these reasons,it was decided to use the Fmoc instead of Boc as the N-protecting group.

[0036] There were two possible synthetic pathways to the targetdipeptide (1), the two amino acids may be coupled first and thenucleobase attached later by the Mitsunobu reaction or the nucleobasemay be incorporated before the peptide coupling. The first approach hasthe advantage of being a more convergent approach. However, apreliminary investigation suggested that it is not satisfactory becauseof the extensive cleavage of the Fmoc group during the Mitsunobureaction. It also seemed likely that displacement of tosylate by anucleobase would give similar premature cleavage of the Fmoc group sincethe reactions require basic conditions.

[0037] A temporary N-protecting group for the hydroxyproline wasrequired, therefore, which is stable to the basic conditions of theMitsunobu reaction but which can be removed, without disturbing thecarboxyl protecting group, in order to allow coupling with Fmoc-glycine(or other amino acid) to give the Fmoc-dipeptide (1). As the carboxylprotecting group must be selectively removed in the presence of the Fmocgroup at the end of the synthesis, an acid-labile protecting groupseemed appropriate. The combination of the acid labile Boc group anddiphenylmethyl (Dpm) ester is ideal because introduction and cleavage ofboth groups are simple and high yielding, The Dpm ester is fullycompatible with the N-Fmoc group and a selective cleavage of a Boc groupin the presence of a diphenylmethyl ester is possible⁸.

[0038] Initial studies were undertaken with the commercially availabletrans-4-hydroxy-L-proline, which was protected as its N-Boc/Dpm esterderivative according to the method described by Tozuka and Takaya.⁹ Thecrystalline derivative (2a) was obtained in greater than 80% yield intwo steps.

[0039] The Mitsunobu reaction on (2a) with N₃-benzoylthymine (BzT) gavethe thymine derivative (3a), together with a less polar product,possibly the O₂-isomer or the elimination product. Fortunately, thethymine derivative (3a) is crystalline and after column chromatographyand one recrystallisation, the pure material was obtained in 51% yield.

[0040] Deprotection of the N-Boc group of the protected thyminederivative (3a) was accomplished with methanolic HCl. The resultingamine salt was reacted with Fmoc-glycine pentafluorophenyl ester in thepresence of diisopropylethylamine (DIEA) to give the protected dipeptide(4a) in excellent yield. Treatment of (4a) with trifluoroacetic acid,either as a neat liquid or in the presence of phenol or anisole as ascavenger,¹⁰ at room temperature for a few hours led to the formation ofroughly equal amounts of two products as shown by tlc and hplc, whichcould not be separated by crystallisation. The unexpected product, whichwas more polar than the desired product, was identified as thedebenzoylated thymine derivative (5a). Since protection of thymine at N₃was only required for selective alkylation at thymine-N₁, thedebenzoylated thymine derivative (5a) was suitable for oligomersynthesis. However, attempts to completely remove the benzoyl group byprolonged treatment with trifluoroacetic acid resulted in a complexmixture. HBr in acetic acid gave better results. Brief treatment of themixture of products from trifluoroacetic acid cleavage with 10% HBr inacetic acid resulted in a complete cleavage of the benzoyl group asshown by hplc. The cleavage conditions have also been applied to thefully protected dipeptide (4a) without pre-treatment withtrifluoroacetic acid with equal success. The synthesis of Fmoc-dipeptidebearing thymine at 4-position in the Cis-L proline series is summarisedin Scheme 1.

[0041] The protected cis- and trans-hydroxy-D-proline (2b) and (2c) wererequired for the preparation of the trans- and cis-D-proline dipeptidesbearing nucleobases. The reaction of N-Boc-cis-4-hydroxy-D-proline withdiphenyidiazomethane gave the Dpm ester (2b) in 90% yield. Inversion ofthe 4-OH group in (2b) to give (2c) was effected by the Mitsunobureaction. By this route, (2c) was prepared in multigram quantities from(2b) in excellent yield (90%, 2 steps) (Scheme 2). The specific rotationof the (2c) ([α]_(D) ²⁵+53.0, c=1.0, EtOH) when compared to that of thetrans-L isomer ([α]_(D) ²⁵−54.3, c=1.0, EtOH) indicated that inversionwas essentially complete.

[0042] The Mitsunobu reaction on the diastereomers, (2b) and (2c), withN₃-benzoylthymine on a 20 mmol scale gave the products (3b) and (3c) in33 and 36% yield respectively. The Boc group in (3b) and (3c) wasremoved with methanolic HCl and the products treated with Fmoc-glycinepentafluorophenyl ester to give the protected dipeptides (4b) and (4c).After treatment with 10% HBr in acetic acid the Fmoc-dipeptide acids(5b) and (5c) were obtained with concomitant cleavage of the N₃-benzoylgroup. The intermediate protected dipeptide (4b) and the final product(5b) were not crystallised as readily as their diastereomers (4c) and(5c). However, the purity of the crude Fmoc-dipeptide (5b) and (5c) wasproved to be satisfactory by hplc.

[0043] The Fmoc-dipeptide (5a), (5b) and (5c) were prepared ingram-quantities for solid phase synthesis. Pentafluorophenyl esters ofthe diastereomeric thymine dipeptides (6a), (6b) and (6c) were allprepared by reactions of the free acids with pentafluorophenol in thepresence of DCCI in dichloromethane.¹¹ These active esters werecrystalline solids which were stable enough to permit purification bysilica gel column chromatography and could be stored for several monthsat −20° C. without apparent decomposition according to ¹H nmr.

[0044] Binding studies between the 10mers derived from coupling of (6a),(6b) and (6c), and poly(dA), showed that the oligomer derived from (6c)binds most strongly. The cis-D proline series was selected therefore forfurther investigation. The protected cis-hydroxy-D-proline (2b) wasconverted into the crystalline trans-D-tosylate (7) in 68% yield by aMitsunobu reaction with methyl p-toluenesulfonate in the presence oftriphenylphosphine and DEAD, according to the method of Peterson andVince.¹² Reactions of (7) and N₆-benzoyladenine in the presence of K₂CO₃and a catalytic amount of 18-crown-6 in DMF afforded the N₉-isomer ofBoc-D-Pro(cis-4-BzA)-ODpm (8) in 42% yield. However, on scaling up, asmall amount of another isomer (5%) was also isolated. This was probablythe N₇-isomer according to the upfield ¹³C chemical shift of adenine C₅(115.0 and 114.6 ppm, rotamers) relative to the major product (123.4ppm)¹³.

[0045] Deprotection of the Boc group in (8) was first attempted bymethanolic HCl as described previously for the thymine derivatives,however, less selectivity was achieved. However, p-toluenesulfonic acidin acetonitrile, which has been successfully applied to deprotect theN-Boc group during the synthesis of cephalosporin derivatives,¹⁴ cleanlyremoved the Boc group without cleaving the Dpm ester. The product wasreacted with Fmoc-glycine pentafluorophenyl ester to gave theFmoc-dipeptide diphenylmethyl ester (9) in 85% yield. Deprotection ofthe Dpm ester with trifluoroacetic acid in the presence of anisole gavethe free acid which was directly converted into the pentafluorophenylester (10) by reacting with pentafluorophenol in the presence of DCCI.The N₆-benzoyl group on adenine remained intact throughout the reactionsequence. Attempted purification of the highly polar pentafluorophenylester (10) by column chromatography found only limited success. However,the crude product after trituration and washing with hexane, was shownby ¹H nmr to contain approximately 10% of dicyclohexylurea (DCU) as theonly contaminant, and was used successfully for solid phase peptidesynthesis.

[0046] Reaction of the trans-D-tosylate (7) with N₄-benzoylcytosine inthe presence of K₂CO₃/18-crown-6 in DMF gave the desired N₁-isomer,Boc-D-Pro(cis-4-N₁-BzC)-ODpm, (11) in 25% yield along with the lesspolar O₂-isomer in 41% yield, which could be readily separated bychromatography on silica gel. The identity of the two isomers wasfurther confirmed by the characteristic downfield shift of the ¹³Cresonance of C_(4 ′) of the O₂-isomer compared to the N₁-isomer. SinceN₄-benzoylcytosine was shown to be partially hydrolysed in hot 85%acetic acid to give uracil,¹⁵ the stability of this group towards acidswas tested before attempting deprotection of the Boc group or thediphenylmethyl ester. The Boc-protected amino acid (11) was treated withtrifluoroacetic acid in the presence of anisole for 2 h. ¹H nmr of theproduct showed that the Boc and Dpm groups were completely removedwhereas the benzoyl group was stable thus demonstrating that thedeprotection conditions were satisfactory.

[0047] Removal of the Boc group of (11) and reaction of the product withFmoc-glycine pentafluorophenyl ester as described for the adenineanalogue gave the protected cytosine dipeptide (12) in 70% overallyield. The benzoylcytosine dipeptide and its pentafluorophenyl ester(13) were synthesised in essentially the same way as the thymine andadenine analogues.

[0048] The Mitsunobu reaction betweenN₂-isobutyryl-O₆-(4′-nitrophenylethyl)guanine¹⁶ and the protectedtrans-4-hydroxy-D-proline (2c) gave the required product, but, could notbe isolated free from diethyl hydrazinedicarboxylate. Treatment with DBUin pyridine to remove the O₆-nitrophenylethyl protecting group followedby column chromatography, however, gave the pure N₉-substitutedisobutyrylguanine derivative (14) as a white solid in 43% overall yield.Removal of the Boc group and reaction of the product with Fmoc-glycinepentafluorophenyl ester gave the protected guanine dipeptide (15) in 52%yield. Removal of the carboxyl protecting group and reaction of theproduct with pentafluorophenol and DCCI gave the isobutyrylguaninedipeptide and its pentafluorophenyl ester (16).

[0049] A model synthesis was first carried out manually on thetrans-D-proline analogue. The target was a T₁₀ cPNA:H-[Gly-D-Pro(trans-4-T)]₁₀-Lys-NH₂. The lysine amide was included at theC-terminus to prevent self-aggregation and to increase water solubility.An acid-labile dimethoxybenzhydrylamine Novasyn-TGR resin was chosen asthe solid support since cleavage with trifluoroacetic acid leadsdirectly to the peptide amide. The polyethyleneglycol matrix alsoimproves the swelling properties of the resin and allows better accessof the reagents to the growing peptide chain. The first lysine residuewas introduced by coupling with Fmoc-Lys(Boc)-OPfp in the presence ofHOBt. The peptide was synthesised from Fmoc-Gly-D-Pro(trans-4-T)—OH(5b), in the presence of HBTU/DIEA according to the standard protocolfor Fmoc-solid phase synthesis.¹⁷ The efficiency of the couplingreactions, which was followed quantitatively after deprotection of theFmoc group by measuring the absorbance of the dibenzofulvene-piperidineadduct (ε₂₆₄=18000) liberated during deprotection, was not as good asexpected despite the use of a large excess of reagents and prolongedreaction times.

[0050] Much better coupling was obtained by first converting (5b) intothe pentafluorophenyl (Pfp) ester (6b) with DCCI and pentafluorophenol,and then performing the coupling in the presence of HOBt. A possiblereason for the low yield in the case of HBTU activation was that theactivated monomer may have been lost by cyclisation to adiketopiperazine derivative. This is a very facile reaction for theactive esters of protected or unprotected dipeptides which containproline at the C-termini, especially in the presence of a base, forexample, Z-Gly-Pro-ONp, is known to spontaneously cyclise under basicconditions.¹⁸ The pentafluorophenyl ester is less reactive than theO-acylisourea or HOBt ester formed during HBTU activation and thecoupling does not require basic conditions, which probably explains theimproved coupling.

[0051] The decathymine chiral peptide nucleic acids with differentstereochemistry at proline (trans-D, cis-D and cis-L), i.e.H-[Gly-D-Pro(trans-4-T)]₁₀-Lys-NH₂, H-[Gly-D-Pro(cis-4-T)]₁₀-Lys-NH₂ andH-[Gly-L-Pro(cis-4T)]₁₀-Lys-NH₂ were successfully prepared by successivecoupling of the corresponding dipeptide pentafluorophenyl estersaccording to the protocol shown in Scheme 3 on 5 μmol scales. Thesyntheses were accomplished rapidly and efficiently. Total amounts ofactivated dipeptides required for each 5 μmol synthesis of a decamerwere approximately 150 mg. The chiral peptide nucleic acids werereleased from the resin and purified according to the standard protocol.In each case, analytical hplc of the crude products showed that theywere 90-95% pure.

[0052] The peptides were purified by reverse phase hplc and theiridentity confirmed by electrospray mass spectrometry (Table 1).Interestingly, these higher oligomers showed an ability to form adductswith alkali metal ions especially potassium in the mass spectrometer, asevidenced by the presence of mass peaks at M+39n, where n is an integralnumber, in addition to the expected molecular ion peak. In some cases,these potassium ion adducts appeared as the major peaks in the massspectra. All the T₁₀ chiral peptide nucleic acids were sufficientlysoluble in water for biological studies (>1 mg/mL at room temperature),although the trans-D-analogue was considerably more soluble than theother two.

[0053] Next the incorporation of different nucleobases into the chiralpeptide nucleic acids was explored. The mixed adenine-thymine peptidenucleic acids of the trans-D and cis-L series were synthesised from thepentafluorophenyi esters without difficulty. However, attempts to removethe nucleobase protecting group (in this case, benzoyl) by treatmentwith aqueous ammonia under various conditions resulted in degradation ofthe peptide as shown by hplc. It seemed unlikely that the degradationresulted from direct hydrolysis or ammonolysis of the peptide bond,since the Gly-Pro and Pro-Gly bonds are stable to hot aqueous ammonia.Hplc and electrospray mass spectral analysis of the degradation productsshowed that they are the dipeptides Gly-Pro (or Pro-Gly) with thenucleobases remaining attached. This suggested that the degradation wasprobably caused by intramolecular attack by the amino group of theN-terminal glycine on the amide carbonyl of the next residue to releasethe bicyclic diketopiperazine, which could undergo further hydrolyticring opening under the deprotection conditions to form the correspondingdipeptide observed in the mass spectrum. The process would be repeateduntil the entire peptide chain was degraded.

[0054] Understanding the mechanism of degradation made it possible toavoid this serious side reaction by modifying the N-terminus of thepeptide nucleic acid in a way that would diminish the nucleophilicity ofthe amino groups. It was therefore decided to find another protectinggroup which could be removed under conditions compatible with thepeptide, preferably without introducing additional steps. The Boc groupwas used as it is labile under the conditions for peptide cleavage fromthe resin but stable under the conditions necessary to deprotect thenucleobases on the solid support. This protection-deprotection schemewas tested by synthesising two mixed A-T sequencesH-[Gly-L-Pro(cis-4-T)]₂-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]₂-Lys-NH₂andH-[Gly-L-Pro(cis-4-T)]₆-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)₂-Lys-NH₂.The fully protected peptides were assembled on the solid support asusual and after the final removal of the N-Fmoc group, the freeN-termini were capped with di-t-butyl dicarbonate (Boc₂O) in thepresence of DIEA in DMF. A qualitative ninhydrin test indicated that thecoupling was essentially complete. After flushing the reaction vesselswith DMF, the resins were treated with 1:1 ethylenediamine-ethanol atroom temperature overnight. This deprotection reagent has been used as amilder alternative to aqueous ammonia for the base labilemethylphosphonate oligonucleotides.¹⁹ The reagent was chosen herebecause the reaction could be carried out at room temperature and in thesame vessel used for the peptide synthesis. Another advantage is thatthe resin swells better in this non-aqueous medium—swelling propertiesof the solid support are is crucial for solid phase reactions. Therelatively non-volatile ethylenediamine and benzamide derivative fromthe cleavage reactions were easily removed by flushing the reactionvessels with DMF. Final cleavage and purification were carried outaccording to the standard method. Reverse phase hplc analysis of thecompletely deprotected peptides showed clean single products in eachcase. The identity of the products was confirmed by mass spectrometry{H-[Gly-L-Pro(cis-4-T)]₂-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]₂-Lys-NH₂:M_(r) calcd. 1832.84, found 1832.40±0.10;H-[Gly-L-Pro(cis-4-T)]₆-[Gly-L-Pro(cis-4-A)-Gly-L-Pro(cis-4-T)]₂-Lys-NH₂:M_(r) calcd. 2945.92, found 2945.32±0.14}.

[0055] The synthesis of chiral peptide nucleic acids incorporating allfour natural nucleobases was undertaken next. The first model sequencesynthesised was the tetramerH-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Lys-NH₂.All the coupling was carried out under the conditions described for theoligothymine peptide nucleic acids. The coupling efficiency wasmonitored by measuring the absorbance of dibenzofulvene-piperidineadduct from the deprotection step and showed that the guanine andcytosine could be introduced efficiently (>90% coupling yield, singlecoupling). The N-terminus of the resin bound peptide was then cappedwith the Boc group after removal of the last N-Fmoc group and then theresin was treated with concentrated aqueous ammonia-dioxane 1:1 at 55°C. overnight to remove the nucleobase protecting groups. Ethylenediaminewas avoided in this instance because it had been shown to causemodification of the cytosine residue in oligonucleotides by displacementof the exocyclic amino group with the aminoethylamino group.²⁰ Finaldeprotection of the Boc group and cleavage from the solid support wascarried out according to the standard protocol. Reverse phase hplcanalysis revealed a single major product which was shown to be thedesired product by electrospray mass spectrometry{H-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Lys-NH₂:M_(r) calcd. 1276.54, found 1277.00±0.07}.

[0056] A decamer mixed-base peptide nucleic acid,H-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-T)-Lys-NH₂,was also synthesised by the standard protocol giving the product (M_(r)calcd. 2973.15, found 2974.80±0.35) in good yield and purity as judgedby reverse phase hplc. The reverse phase hplc chromatogram andelectrospray mass spectrum of the decamer are shown in FIG. 1 and FIG.2.

[0057] Hybridization Studies

[0058] The deca-thymine glycyl-proline peptide nucleic acids with cis-L,trans-D and cis-D stereochemistry, and C-terminal L-lysinamide weremixed in 1:1 ratio with poly(rA) and poly(dA) in the presence of 150 mMNaCl and sodium phosphate buffer (10 mM Na⁺, pH 7.0) and the Tmdetermined (FIG. 3). The cis-D- and cis-L-PNAs, but not the trans-D-PNAshowed well defined single-transition melting curves with both poly(rA)and poly(dA). The magnitudes of absorbance change were of the order of3040%. The Tm were as shown in Table 2.

[0059] The slightly higher Tm for the chiral peptide nucleicacid.poly(rA) complexes suggests that they are slightly more stable thanthe peptide nucleic acid.poly(dA) complexes. Although both cis-D andcis-L analogues gave melting curves with poly(dA) and poly(rA), only thecis-D analogue gave a well-defined melting curve with (dA)₁₀, withT_(m)=61° C., under the same conditions. The cis-L analogue gave a broadmelting curve with a T_(m) near room temperature. These results suggestthat there is a stronger interaction between the cis-D analogue, whichpossessed the same absolute stereochemistry as natural oligonucleotides.

[0060] Further investigation of the nature of the cis-D peptide nucleicacid-oligonucleotide interaction was undertaken by determining themelting curve with (dT)₁₀. The 1:1 mixture with (dT)₁₀ showed nosignificant increase in absorbance at 260 nm on heating whereas the 1:1mixture with (dA)₁₀ showed strong hyperchromicity (ca. 20%) suggestingthat the binding is probably specific for A.T pairs, presumably byWatson-Crick base pairing.

[0061] In order to determine the stoichiometry of the peptide nucleicacid-nucleic acid complexes, a titration experiment betweencis-D-stereomer and poly(rA) was investigated. A well-defined mixingcurve was obtained with minima at 1:1 ratio of peptide nucleic tonucleic acid in sodium phosphate buffer (10 mM Na⁺, pH 7.0) suggesting a1:1 stoichiometry (FIG. 4). A similar titration experiment with (dA)₁₀under the same conditions gave essentially the same result.

[0062] All four bases found in DNA were introduced into theglycyl-proline building units with the cis-D configuration and fromthese mixed cPNAs containing all four nucleobases have been made. Thesequence GTAGATCACT, capped at its C-terminus with L-lysinamide wassynthesised, and its binding properties with oligonucleotidesinvestigated. Since it is important to determine the preferredorientation of binding of these novel cPNAs to oligonucleotides, both ofthe possible complementary oligonucleotides were prepared, i.e.5′-CATCTAGTGA-3′ and 5′-AGTGATCTAC-3′ and hybridised with the chiralPNA. Their Tm values were 47° C. and 43° C. respectively indicating thatthe N-terminus of the cPNA preferentially binds to the 5′-terminus ofthe oligonucleotide, and the C-terminus to the 3′-terminus of theoligonucleotide. This is known as the antiparallel mode of binding , butit is seen that the stability of the alternative parallel bindingcomplex is only slightly less stable.

[0063] Following the promising results obtained on the binding isstudies of the cPNA with complementary oligonucleotides by Tmmeasurement, a ¹H NMR experiment was performed on the mixed sequencedecamer,H-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-T)-LysNH₂,and its complementary oligonucleotides, both in parallel andantiparallel fashion. Unfortunately upon mixing the two components atthe mmolar concentration required, a white precipitate formedimmediately and no NMR signals could be observed apart from those ofexcess starting material. It is clear that even though the cPNA and theoligonucleotide alone are freely soluble in water, the complex formedbetween them is not at the high concentration required. In an attempt toovercome this problem it was decided to synthesise a cPNA analogue,containing a hydrophilic spacer amino acid—namely serine—in place of theglycine spacer in the backbone while retaining the cis-D-configurationof the proline moiety. As there are two enantiomers of serine and ourpreliminary molecular model suggested that the stereochemistry of thespacer amino acid may have considerable effects on the binding strengthof the resulting diastereomeric cPNAs with A- and B-forms of DNA, bothenantiomers of serine were studied. Thus the target molecules werediastereomeric thymine-decamers H-[L-Ser-D-Pro(cis-4-T)]₁₀LysNH₂(“LD-ST10”) and H-[D-Ser-D-Pro(cis-4-T)]₁₀LysNH₂ (“DD-ST10”).

[0064] The required Fmoc-protected dipeptide diastereoisomeric synthons(17) were synthesised from Boc-D-Pro(cis-4-N³BzT)-Odpm (3c) in ananalogous manner to the glycylproline analogue. The serine hydroxyl sidechain was protected as a t-butyl ether as in the traditionalFmoc-O^(t)Bu orthogonal protection scheme. The Boc- group in thestarting material was removed by p-TsOH in acetonitrile as describedpreviously, the amine tosylate was then reacted with Fmoc-Ser(OtBu)—OHin the presence of DCCI and HOBt in MeCN/DMF, after neutralisation withDIEA. Both enantiomers of serine gave similar yields (70-90%) of thedesired protected dipeptides, Fmoc-L-Ser(O^(t)Bu)-D-Pro-(cis-4-BzT)-ODpmand Fmoc-D-Ser(O^(t)Bu)-D-Pro-(cis-4-BzT)-ODpm, which were isolated aswhite amorphous solids after column chromatography and werecharacterised by ¹H, ¹³C NMR and APCI-MS. Deprotection of the ODpm esterof Fmoc-L-Ser(O^(t)Bu)-D-Pro-(cis-4-BzT)-ODpm was found to beproblematic since catalytic transfer hydrogenolysis using differenthydrogen donors including ammonium formate, formic acid and cyclohexeneand catalysts—Pd black, 10% Pd/C, and freshly prepared 5% Pd/BaSO₄, gaveunsatisfactory results. Acidic conditions were an alternative, ifselectivity between the Dpm ester and t-Bu ether could be achieved.Various combinations were attempted, but 4M HCl in dioxane appeared togive the best result. The deprotected material, consisting a mixture ofthe desired Fmoc-dipeptide acid and some debutylated product wassubjected to a reaction with pentafluorophenol/DCCI to give the finalactive ester of the Fmoc-dipeptide, which could be purified by columnchromatography on silica gel (Scheme 4). The final products wereobtained in a pure form and were characterised by ¹H NMR and APCI-MS. Itshould be noted that the benzoyl protecting group on the thymine ringremained intact throughout the reaction sequence but this should not bea problem since this benzoyl group is removed readily upon treatmentwith 20% piperidine in DMF during deprotection of the Fmoc group in thesolid phase synthesis step.

[0065] Solid phase synthesis of the two cPNA decamers, LD-ST10 andDD-ST10, were carried out according to our standard protocol on a 5 μmolscale and the efficiency of each coupling step was monitored bymeasuring the absorbance of the dibenzofulvene-piperidine adductreleased from deprotection of the Fmoc group and this showed that thecoupling proceeded efficiently (95-100%). Both cPNA diastereomers couldbe purified to give the pure 10-mers by HPLC which gave correct massesby ESI-MS (3266, M-H+K, identical spectra for both isomers). Thepurified cPNAs, LD-ST10 and DD-ST10, were obtained in 28 and 16% yieldrespectively. Both of the serine-containing cPNAs were freely soluble inwater at a concentration of 2 mM.

[0066] A ¹H NMR study of a mixture of DD-ST10 and dA₁₀ was attempted.Initially the ¹H NMR spectra of both components were recorded separately(at a concentration of 0.53 mM for the DD-ST1 0 and 0.67 mM for the dA₁₀in 10% D₂O in H₂O), which showed the expected resonances. On addition of20 mol % of dA₁₀ (as a concentrated aqueous solution) to a solution of0.5 mM of DD-ST10 in 10% D₂O-H₂O, an immediate precipitation occurred,and it was clear that a structure of the complex in solution could notbe determined.

[0067]FIG. 1: Reverse phase hplc chromatogram of the crude decamerpeptide nucleic acidH-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-T)-Lys-NH₂;Inset: Hplc chromatogram of the purified peptide nucleic acid underidentical conditions; Hplc conditions: μBondapak C-18 reverse phase hplccolumn; solvents: A=0.1% TFA in acetonitrile B=0.1% aqueous TFAisocratic A:B 10:90; flow rate 1.5 ml/min; detection wavelength 260 nm.

[0068]FIG. 2: Electrospray mass spectrum of the purified peptide nucleicacid decamer H-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-G)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-T)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-A)-Gly-D-Pro(cis-4-C)-Gly-D-Pro(cis-4-T)-Lys-NH₂.The potassium adducts [M_(r)=3012 (M⁺K⁺−H⁺) and 3051 (M+2K⁺−2H⁺)] werealso observed in addition to the product (M_(r)=2974).

[0069]FIG. 3. The melting curves of the 1:1 hybrids of (a)H-[Gly-L-Pro(cis-4-T)]₁₀-Lys-NH₂, (b) H-[Gly-D-Pro(cis-4-T)]₁₀-Lys-NH₂,(c) H-[Gly-D-Pro(trans-4-T)]₁₀-Lys-NH₂ with poly(dA) at 150 mM NaCl, 10mM sodium phosphate, pH 7.0 and (d) poly(dA) alone. The poly(dA)concentration was 10.8 μM dA nucleotide. The melting curves wererecorded at 260 nm and the rate of heating was 0.5° C./min.

[0070]FIG. 4. (Titration of the chiral PNAH-[Gly-D-Pro(cis-4-T)]₁₀-Lys-NH₂ with poly(rA) at 20” C. Calculatedamounts of poly(rA) in 10 mM sodium phosphate buffer pH 7.0 (0.282 mM rAnucleotide) were added to a solution of 16.5 μMH-[Gly-D-Pro(cis-4-T)]₁₀-Lys-NH₂ in the same buffer. The ratio ofobserved OD₂₆₀ to calculated OD₂₆₀ is plotted against % mol ofH-[Gly-D-Pro(cis-4-T)]₁₀-Lys-NH₂.

[0071] Experimental

[0072] Melting points were recorded on a Kofler block apparatus and arequoted uncorrected. Specific rotations were measured on a Perkin-Elmer241 polarimeter. IR spectra were recorded on a Perkin-Elmer 1750 FourierTransform Infrared spectrometer. Elemental analyses were performed on aCarlo Erba CHN analyser model 1106.

[0073] Routine ¹H and ¹³C nmr spectra were recorded on a Varian Gemini200 spectrometer operating at 200 MHz (¹H) and 50.28 MHz (¹³C). ¹³Cspectra were recorded in broad band decoupled mode and the chemicalshift assignment was assisted by a DEPT experiment performed on theVarian Gemini 200 spectrometer. High field ¹H nmr were recorded on aBruker AMX 500 spectrometer (500 MHz). ¹⁹F nmr spectra were recorded ona Bruker AM 250 at 235.35 MHz. ¹H and ¹³C chemical shifts are quoted inppm relative to tetramethylsilane and were internally referenced to theresidual protonated solvent signal. ¹⁹F chemical shifts were externallyreferenced to CFCl₃ in CHCl₃.

[0074] Chemical ionisation and fast atom bombardment mass spectra wererecorded on a VG 20-250 masslab and a VG Micromass ZAB-1 F massspectrometer. Electrospray mass spectra were recorded on a VG BiotechBioQ or VG Biotech Platform. Masses are quoted as m/z unless otherwisestated, only the molecular ions and major fragments being quoted.

[0075] Distilled water was used for all chemical experiments. Chemicalsand solvents were obtained from Aldrich Chemical Company Ltd., AvocadoResearch Chemicals Ltd. and Lancaster Synthesis Ltd. and were purifiedaccording to the literature,²¹ if necessary. N-Boc-trans-4-hydroxy-L-proline and Fmoc-glycine pentafluorophenyl ester wereobtained from Calbiochem-Novabiochem Ltd. p-Toluenesulfonyl chloride waspurified by recrystallisation from petroleum ether (b.p. 60-80° C.). DMFwas peptide synthesis grade obtained from Rathburn Chemical Ltd. and wasused without further purification except when strictly anhydrousconditions were required where it was re-distilled from calcium hydrideunder reduced pressure. Acetonitrile was hplc grade obtained fromRathburn and used without further purification. THF and dioxane weredistilled from sodium wire/benzophenone under argon and stored over 4Amolecular sieve. Pyridine was distilled from calcium hydride and storedover 4A molecular sieve. Moisture-sensitive reactions were performedunder argon in flame-dried glassware.

[0076] N-tert-Butoxycarbonyl-trans-4-hydroxy-L-proline DiphenylmethylEster (2a) and N-tert-butoxycarbonyl-cis-4-hydroxy-D-prolinediphenylmethyl ester (2b)

[0077] To a solution of freshly prepared diphenyldiazomethane²² (3.50 g,18.0 mmol) in ethyl acetate (20 ml) was addedN-Boc-trans-4-hydroxy-L-proline (3.25 g, 14.0 mmol) in ethyl acetate (30ml). Nitrogen gas was slowly evolved from the solution and the intensepurple colour of diphenyidiazomethane was gradually discharged. Thesolution was stirred at room temperature overnight using a CaCl₂ guardtube. Evaporation of the solvent followed by precipitation of theproduct from ethyl acetate-petroleum ether b.p. 40-60° C. gave a whitesolid, N-tert-butoxycarbonyl-trans-4-hydroxy-L-proline diphenylmethylester (2a), (5.10 g, 91%), m.p. 93-95° C. (lit.⁹ m.p. 103-104° C.),δ_(H) (200 MHz; CDCl₃) 1.22 and 1.47 (9H, 2xs, Boc rotamers), 1.95-2.50[3H, br m, CH ₂(3) and OH], 3.45-3.74 [2H, br m, CH ₂(5)], 4.40-4.65[2H, br m, CH(2)and CH(4)], 6.95 (1H, br s, CHPh₂), 7.25-7.55 (10OH, brm, phenyl CH); ν_(max) (KBr)/cm⁻¹ 3491br (O—H), 1728s (C═O ester), 1693s(C═O urethane); m/z (ES MS) 436 (M+K⁺, 55%), 420 (M+Na⁺, 100), 415(M+NH₄ ⁺, 15), 398 (M+H⁺, 72); [α]_(D) ²⁵ −54.3 (c=1.0, EtOH).

[0078] The cis-D diastereoisomer was prepared similarly starting fromN-Boc-cis-hydroxy->proline (11.6 g, 50.0 mmol) and diphenyidiazomethane(11.3 g, 58.0 mmol) in ethyl acetate (150 ml).N-tert-Butoxycarbonyl-cis-4-hydroxy-D-proline diphenylmethyl ester (2b)was obtained as a white solid after precipitation from ethylacetate-petroleum ether b.p. 40-60° C. (17.9 g, 90%) m.p. 102-105° C.,(Found C, 69.5; H, 6.8; N, 3.3%; C₂₃H₂₇NO₅ requires C, 69.5; H, 6.8; N,3.5%), δ_(H) (200 MHz; CDCl₃) 1.26 and 1.30 (9H, 2xs, Boc rotamers),2.08 and 2.35 [2H, m, CH ₂(3)], 2.88 and 3.09 (1H, 2xd, J=9.6 Hz, OHrotamers), 3.57-3.66 [2H, br m, CH ₂(5)], 4.32 [2H, m, CH(4)], 4.42-4.58[1H, m, CH(2)] 6.91 and 6.99 (1H, 2xs, CHPh₂ rotamers), 7.25-7.48 (10OH,br m, phenyl CH); δ_(C) (50.28 MHz; CDCl₃) 28.0 and 28.3 (Boc CH₃rotamers), 37.6 and 38.6 [CH₂(3) rotamers], 55.4 and 55.9 [CH₂(5)rotamers], 58.0 and 58.1 [CH(2) rotamers], 70.1 and 71.2 [CH(4)rotamers], 78.1 and 78.6 (CHPh₂ rotamers), 80.4 and 80.6 (Boc C),127.2-128.8 (phenyl CH rotamers), 139.6 and 139.8 (phenyl C rotamers),154.1 (Boc CO), 174.0 (ester CO rotamers); ν_(max) (KBr)/cm⁻¹ 3466br(O—H), 1749s (C═O ester), 1687s (C═O urethane); [α]_(D) ²⁵+41.2 (c=1.0,EtOH).

[0079] N-tert-Butoxycarbonyl-trans-4-hydroxy-D-proline DiphenylmethylEster (2c)

[0080] N-Boc-cis-hydroxy-D-proline diphenylmethyl ester (2b)(0.20 g,0.50 mmol), triphenylphosphine (0.160 g, 0.60 mmol) and formic acid (25μl, 0.65 mmol) were dissolved in dry THF (10 ml) and cooled in an icebath. DEAD (100 gL, 0.60 mmol) was added dropwise. The reaction mixturewas stirred under nitrogen at room temperature overnight. The solventwas evaporated and the residue was chromatographed on silica gel usingdichloromethane:acetone 20:1 as eluant to give the 4-formate ester(R_(f)=0.50) as a colouriess oil (0.248 g, quant.), δ_(H) (200 MHz;CDCl₃) 1.25 and 1.47 (9H, 2xs, Boc rotamers), 2.10-2.55 [2H, br m, CH₂(3)], 3.57-3.80 [2H, br m, CH ₂(5)], 4.48-4.64 [1H, m, CH(2)],5.35-5.43 [1H, br m, CH(4)], 6.91 and 6.95 (1H, 2xs, CHPh₂ rotamers),7.25-7.42 (10H, br m, phenyl CH), 8.03 [1H, s, HC(O)].

[0081] The oil was taken up in methanol (10 ml) and concentrated aqueousammonia (d 0.880; 0.5 ml) was added. Tic analysis indicated completereaction after stirring at room temperature for 1 h. The solvent wasremoved under reduced pressure and the residue chromatographed on silicagel column using diethyl ether as eluant to give the product (R_(f)0.30) as a white foam (0.179 g, 90% from 2b), which was further purifiedby reprecipitation from ethyl acetate-petroleum ether (b.p. 40-60 ” C)to give N-tert-butoxycarbonyl-trans-4-hydroxy-D-proline diphenylmethylester (2c) as a white solid, m.p. 105-108° C., (Found C, 69.8; H, 6.9;N, 3.3%; C₂₃H₂₇NO₅ requires C, 69.5; H, 6.8; N, 3.5%), δ_(H) (200 MHz;CDCl₃) 1.22 and 1.47 (9H, 2xs, Boc rotamers), 2.02 and 2.30 [2H, 2x brm, CH ₂(3)], 2.70 and 2.82 (1H, 2xbrd, J=3.1 and 3.4 Hz, OH rotamers),3.45-3.70 [2H, br m, CH ₂(5)], 4.42-4.65 [2H, br m, CH(2)and CH(4)],6.90 and 6.95 (1H, 2xs, CHPh₂), 7.25-7.45 (10H, br m, phenyl CH); δ_(C)(50.28 MHz; CDCl₃) 27.9 and 28.3 (Boc CH₃ rotamers), 38.1 and 38.9[CH₂(3) rotamers], 54.7 [CH₂(5)], 57.8 and 58.1 [CH(2) rotamers], 69.2and 70.0 [CH(4) rotamers], 77.2 and 77.6 (CHPh₂ rotamers), 80.6 (Boc C),127.1-128.8 (phenyl CH rotamers), 140.0 and 140.2 (phenyl C rotamers),154.4 (Boc CO), 172.0 (ester CO); ν_(max) (KBr)/cm⁻¹ 3491br (O—H), 1728s(C═O ester), 1693s (C═O urethane); [α]_(D) ²⁵+53.0 (c=1.0, EtOH).

[0082] N-tert-Butoxycarbonyl-trans-4-(p-toluenesulfonyloxy)-D-prolineDiphenylmethyl Ester (7)

[0083] A solution of N-Boc-cis-4-hydroxy-D-proline diphenylmethyl ester2b) (6.40 g, 16.1 mmol), triphenylphosphine (4.50 g, 16.8 mmol) andmethyl-p-toluenesulfonate (3.04 g, 16.4 mmol) in THF was treated withDEAD (2.80 ml, 4.2 mmol) dropwise at −78° C. The reaction was allowed togradually warm to room temperature and the stirring continued overnight.Evaporation followed by column chromatography (SiO₂, diethyl ether,R_(f) 0.61) gave the crude product as an oil which was re-precipitatedfrom diethyl ether-petroleum ether b.p. 40-60° C. to give theessentially pure product as a white solid (6.01 g, 68%).Recrystallisation from ethyl acetate-petroleum ether (b.p. 40-60° C.)gave N-tert-butoxycarbonyl-trans-4-(p-toluenesulfonyloxy)-D-prolinediphenylmethyl ester (7) as white crystals, m.p. 147-149° C., (Found C,65.2; H, 5.8; N, 2.4%; C₃₀H₃₃NO₇S requires C, 65.3; H, 6.0; N, 2.5%),δ_(H) (200 MHz; CDCl₃) 1.22 and 1.44 (9H, 2xs, Boc rotamers), 1.89-2.18and 2.32-2.66 [2H, m, CH₂(3)], 2.46 (3H, s, tosyl CH ₃), 3.54-3.74 [2H,m, CH ₂(5)], 4.50 [1H, m, CH(2)], 4.98 [1H, br m, CH(4)], 6.87 and 6.92(1H, 2xs, CHPh₂ rotamers), 7.25-7.38 (12H, m, aromatic CH), 7.77 (2H, d,J=8.2 Hz, tosyl CH); δ_(C) (50.28 MHz; CDCl₃) 21.6 (tosyl CH ₃), 27.9and 28.2 (Boc CH ₃ rotamers), 35.6 and 37.0 [CH ₂(3) rotamers], 51.8 and52.1 [CH ₂(5) rotamers], 57.4 and 57.6 [CH(2) rotamers], 78.3 and 78.9(CH(4) rotamers], 78.2 and 78.7 (CHPh₂ rotamers), 80.7 and 80.9 (Boc Crotamers), 127.0-130.3 (aromatic CH), 133.5, 139.9 and 145.3 (aromaticC), 154.6 and 154.8 (Boc CO rotamers), 171.3 (ester CO rotamers);ν_(max) (KBr)/cm⁻¹ 1742s (C═O ester), 1704 (C═O urethane), 1402s(—SO₂O—), 1174s (—SO₂O—).

[0084] N-tert-Butoxycarbonyl-4-(N₃-benzoylthymin-1-yl)prolineDiphenylmethyl Esters (3a), (3b) and (3c)

[0085] N-Boc-trans-4-hydroxy-L-proline diphenylmethyl ester (2a) (0.425g, 1.07 mmol), triphenylphosphine (0.290 g, 1.10 mmol) andN₃-benzoylthymine (0.250 g, 1.09 mmol) were dissolved in dry THF (10 ml)and the solution was cooled to −15° C. DEAD (180 μL, 1.10 mmol) was thenadded dropwise with stirring. The reaction mixture was stirred underargon at room temperature overnight. The solvent was evaporated and theresidue was chromatographed on silica gel using dichloromethane-acetone20:1 as eluant to giveN-tert-butoxycarbonyl-cis-4-(N₃-benzoylthymin-1-yl)-L-prolinediphenylmethyl ester (3a) (R_(f) 0.56), which was recrystallised fromethanol to give a white fluffy solid (0.310 g, 51%), m.p. 183-185° C.,(Found C, 68.9; H, 5.5; N, 6.7%; C₃₅H₃₅N₃O₇ requires C, 69.0; H, 5.8; N,6.9%), δ_(H) (200 MHz; CDCl₃) 1.30 and 1.49 (9H, 2xbr s, Boc rotamers),1.82 (3H, br s, thymine CH ₃), 2.05 and 2.85 [2H, br m, CH ₂(3′)], 3.65(1H, br m) and 4.02 (1H, dd, J=12.0, 8.0 Hz)[CH ₂(5′)], 4.54 [1H, br m,CH(2′)], 5.26 [1H, br m, CH(4′)], 6.94 (1H, s, CHPh₂), 7.12 and 7.18[1H, 2xbr s, CH(6) rotamers], 7.30-7.42 (10H, br m, phenyl CH), 7.50(2H, t, J=7.9 Hz, benzoyl m-CH), 7.67 (1H, t, J=7.0 Hz, benzoyl p-CH),7.92 (2H, d, J=7.0 Hz, benzoyl o-CH); δ_(C) (50.28 MHz; CDCl₃) 12.3(thymine CH₃), 27.9 and 28.2 (Boc CH₃ rotamers), 34.9 and 35.3 [CH₂(3′)rotamers], 49.3 and 49.4 [CH₂(5′) rotamers], 52.1 and 52.5 [CH(4′)rotamers], 57.5 [CH₂(2′)], 78.1 and 78.4 (CHPh₂ rotamers), 81.3 (Boc C),111.7 [C(5)], 126.9-130.6 (aromatic CH), 131.6 (benzoyl C), 135.3(benzoyl p-CH), 136.0 and 136.2 [CH(6) rotamers], 139.4 (phenyl C),150.1 [C(2)], 153.6 (Boc CO), 162.6 [C(4)], 169.1 (benzoyl CO), 171.6(ester CO); m/z (FAB+) 632 (M+Na⁺, 10%), 610 (M+H⁺, 39), 554([M—C₄H₈+H]⁺, 27), 506 ([M—PhCO+H]⁺, 18), 338 ([M—PhCO—Ph₂CH]⁺, 20), 266(31), 231 (BzT+H⁺, 52), 167 (Ph₂CH⁺, 100), 105 (PhCO⁺, 24), 57 (C₄H₉ ⁺,28); ν_(max) (KBr)/cm⁻¹ 1751, 1694 and 1659 (C═O); [α]_(D) ²⁵ −17.2(c=1.0, DMF).

[0086] N-tert-Butoxycarbonyl-trans-4-(N₃benzoylthymin-1-yl)-D-prolinediphenylmethyl ester (3b) was similarly prepared starting from the cis-Dalcohol (2b) (8.0 g, 20 mmol). The product was obtained as a white solidafter column chromatography (SiO₂, dichloromethane-acetone 20:1) andtrituration with diethyl ether (4.20 g, 33%). Recrystallisation fromethyl acetate-hexane gave an analytically pure sample as white crystals,m.p. 189-192° C., (Found C, 68.9; H, 5.5; N, 6.7%; C₃₅H₃₅N₃O₇ requiresC, 69.0; H, 5.8; N, 6.9%), δ_(H) (200 MHz; CDCl₃) 1.30 and 1.47 (9H,2xs, Boc rotamers), 1.80 (3H, s, thymine CH ₃),2.40 and 2.58 [2H, m, CH₂(3′)], 3.55-4.02 [2H, m, CH ₂(5′)], 4.53-4.64 [1H, m, CH(2′)], 5.12[1H, m, CH(4′)], 6.90 and 6.94 (1H, 2xs, CHPh₂ rotamers), 7.00 [1H, s,CH(6) rotamers], 7.35-7.45 (10H, br m, phenyl CH), 7.52 (2H, t, J=8.0Hz, benzoyl m-CH), 7.69 (1H, t, J=7.2 Hz, benzoyl p-CH), 7.94 (2H, J=7.2Hz, benzoyl o-CH); δ_(C) (50.28 MHz; CDCl₃) 12.6 (thymine CH₃), 27.9 and28.2 (Boc CH₃ rotamers), 33.4 and 35.1 [CH₂(3′) rotamers], 49.1 and 49.5[CH₂(5′) rotamers], 53.8 and 54.4 [CH(4′) rotamers], 57.7 and 58.0[CH₂(2′) rotamers], 77.9 and 78.2 (CHPh₂ rotamers), 81.2 and 81.3 (Boc Crotamers), 111.9 [C(5)], 127.0-130.6 (aromatic CH), 131.6 (benzoylC),135.3 (benzoyl p-CH), 136.2 [CH(6)], 139.4 and 139.7 (phenyl Crotamers), 149.9 [C(2)], 153.6 (Boc CO), 162.7 [C(4)], 169.1 (benzoylCO), 171.0 (ester CO); ν_(max) (KBr)/cm⁻¹ 1739s (C═O), 1703s (C═O),1664s (C═O); [α]_(D) ²⁵ _(11.3) (c=1.03, DMF).

[0087] N-tert-Butoxycarbonyl-cis-4-(N₃-benzoylthymin-1-yl)-D-prolinediphenylmethyl ester (3c) was similarly prepared starting from thetrans-D alcohol (2c) (7.62 g, 19.2 mmol). The product was obtained as awhite crystalline solid after column chromatography (SiO₂,dichloromethane-acetone 20:1) and recrystallisation from ethanol (4.20g, 36%), m.p. 183-186° C., (Found C, 69.1; H, 5.8; N, 6.8% C; C₃₅H₃₅N₃O₇requires C, 69.0; H, 5.8; N, 6.9%), δ_(H) (200 MHz; CDCl₃) 1.30 and 1.49(9H, 2xbr s, Boc rotamers), 1.81 (3H, br s, thymine CH ₃), 2.04 and 2.86[2H, 2xbr m, CH ₂(3′)], 3.66 (1H, br m) and 4.02 (1H, dd, J=12.0, 8.0Hz) [CH ₂(5′)], 4.53 [1H, br m, CH(2′)], 5.26 [1H, br m, CH(4′)], 6.95(1H, s, CHPh₂), 7.12 and 7.18 [1H, 2xbr s, CH(6) rotamers], 7.30-7.44(10H, br m, phenyl CH), 7.50 (2H, t, J=7.9 Hz, benzoyl m-CH), 7.67 (1H,t, J=7.0 Hz, benzoyl p-CH), 7.92 (2H, d, J=7.0 Hz, benzoyl o-CH);ν_(max) (KBr)/cm⁻¹ 1751s (C═O), 1699s (C=O), 1661s (C═O); [α]_(D) ²⁵+16.9 (c=1.03, DMF).

[0088] N-tert-butoxycarbonyl-cis-4-(N₆-benzoyladenin-9-yl)-D-prolineDiphenylmethyl Ester (8)

[0089] A mixture of the trans-D tosylate (7) (0.552 g, 1.00 mmol),N₆-benzoyladenine (0.595 g, 2.50 mmol), anhydrous K₂CO₃ (0.700 g, 5.00mmol) and 18-crown-6 (0.100 g) in DMF (5 ml) was stirred under argon at80° C. overnight. The reaction mixture was diluted with dichloromethane(20 ml) and washed with water, dried (MgSO₄) and evaporated to give thecrude product, which was purified by column chromatography (SiO₂, 2.5%methanol in dichloromethane, R_(f) 0.27) to give the product as a whitefoam (0.260 g, 42%) which was spectroscopically pure. Furtherrecrystallisation from ethanol-water gave an analytically pureN-tert-butoxycarbonyl-cis-4-(N₆benzoyladenin-9-yl)-D-prolinediphenylmethyl ester (8) as colourless needles, m.p. 115-119° C., (FoundC, 65.9; H, 5.5; N, 13.1%; C₃₅H₃₄N₆O₅.H₂O requires C, 66.0; H, 5.7; N,13.2%), 5H (200 MHz; CDCl₃) 1.31 and 1.49 (9H, 2xs, Boc rotamers), 2.52and 2.90 [2H, 2xbr m, CH ₂(3′)], 3.90-4.20 [2H, br m, CH2(5′)], 4.52 and4.63 [1H, 2xbr m, CH(2′) rotamers], 5.14 [1H, br m, CH(4′)], 6.83 (1H,s, CHPh₂), 7.15-7.28 (10H, m, phenyl CH), 7.35-7.60 (m, 3H, benzoyl m-and p-CH), 7.95-8.05 [3H, m, CH(8) and benzoyl o-CH], 8.68 [1H, s,CH(2)], 9.39 (1H, s, NH); δ_(C) (50.28 MHz; CDCl₃) 28.0 and 28.2 (BocCH₃ rotamers), 34.5 and 35.7 [CH₂(3′) rotamers], 49.9 and 50.5 [CH₂(5′)rotamers], 52.3 and 52.8 [CH(4′) rotamers], 57.6 [CH(2′)], 77.8 (CHPh₂),81.3 (Boc C), 123.4 [C(5)], 127.0-129.0 (aromatic CH), 132.9 (aromaticCH), 133.9 (aromatic C), 139.4 and 139.5 (aromatic C),141.5 [CH(8)],149.8 [C(4)], 152.0 [C(6)], 152.7 [CH(2)], 153.6 and 154.0 (Boc COrotamers), 165.1 (benzoyl CO), 170.9 (ester CO); m/z (ES MS) 619 (M+H⁺,100%); ν_(max) (KBr)/cm⁻¹ 1748 (C═O), 1697s (C═O); λ_(max) (CHCl₃)/nm285 (ε/dm³.mol⁻¹.cm⁻¹ 2.1×10⁴); [α]_(D) ²⁵ +14.1 (c=0.63, CHCl₃).

[0090] N-tert-Butoxycarbonyl-cis-4-(N₄-benzoylcytosin-1-yl)-D-prolineDiphenylmethyl Ester (11) andN-tert-butoxycarbonyl-cis-4-(4-benzoylaminopyrimidin-2-oxy)-D-prolineDiphenylmethyl Ester

[0091] A reaction mixture containing the trans-D tosylate (7) (1.10g,2.00 mmol), N₄-benzoylcytosine (0.475 g, 2.20 mmol), anhydrous K₂CO₃(0.300 g, 2.20. mmol) and 18-crown-6 (200 mg) in DMF (10 ml) was stirredat 70-80° C. under argon overnight. The white suspension was dilutedwith dichloromethane (75 ml), filtered through celite and the organicphase washed with water. Evaporation gave the crude product as an oilwhich was purified by column chromatography (SiO₂, ethyl acetate). Themore polar fractions (R_(f) 0.33) were combined and evaporated to givethe N₁-isomer (0.299 g, 25%) as a white foam. Recrystallisation fromethanol-water gave white crystals ofN-tert-butoxycarbonyl-cis-4-(N₄-benzoylcytosin-1-yl)-D-prolinediphenylmethyl ester (11), m.p. 133-135° C., (Found C, 65.8; H, 6.5; N,8.8%; C₃₄H₃₄N₄O₆.C₂H₅OH.H₂O requires C, 65.6; H, 6.4; N, 8.5%), 5H (200MHz; CDCl₃) 1.30 and 1.49 (9H, 2xs, Boc rotamers), 2.20 and 2.90 [2H, brm, CH ₂(3′)], 3.50-3.80 and 3.95-4.15 [2H, 2xbr m, CH ₂(5′)], 4.45-4.70[1H, br m, CH(2′)], 5.28 [1H, br m, CH(4′)], 6.87 (1H, s, CHPh₂),7.15-7.40 (10H, m, phenyl CH), 7.40-7.75 15H, m, CH(5), CH(6) andbenzoyl m- and p-CH], 7.89 (2H, d, J=7.4 Hz, benzoyl o-CH), 8.83 (1H, brs, NH); δ_(C) (50.28 MHz; CDCl₃) 27.7 and 28.0 (Boc CH₃ rotamers), 34.4and 36.0 [CH₂(3′) rotamers], 49.6 and 50.5 [CH₂(5′) rotamers], 54.3 and54.9 [CH(4′) rotamers], 57.6 [CH(2′)], 78.0 and 78.3 (CHPh₂ rotamers),81.3 (Boc C), 96.8 [CH(5)], 127.0-129.2 (aromatic CH), 133.4 (benzoylC), 139.5 (aromatic C), 145.2 and 145.7 [CH(6) rotamers], 153.8 (Boc COrotamers), 155.6 [C(2)], 162.0 [C(4)], 166.8 (benzoyl CO), 171.3 (esterCO); m/z (ES MS) 595 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1743 (C═O), 1704s(C═O); λ_(max) (CHCl₃)/nm 266 (ε/dm³.mol⁻¹.cm⁻¹ 8.9×10⁴), 312 (3.4×10⁴);[α]_(D) ²³ −13.6 (c=0.50, CHCl₃).

[0092] The less polar fractions (R_(f) 0.61) were combined andre-chromatographed (SiO₂, dichloromethane:acetone 10:1) to give theO₂-isomer as a white foam (0.489 g, 41%) which was recrystallised fromethanol to give colourless needles ofN-tert-butoxycarbonyl-cis-4-(4-benzoylaminopyrimidin-2-oxy)-D-prolinediphenylmethyl ester m.p. 145-147° C., (Found C, 68.6; H, 5.5; N, 9.4%;C₃₄H₃₄N₄O₆ requires C, 68.7; H, 5.8; N, 9.4%). δ_(H) (200 MHz; CDCl₃)1.27 and 1.46 (9H, 2xs, Boc rotamers), 2.42 and 2.63 [2H, br m, CH₂(3′)], 3.60-4.20 [2H, br m, CH ₂(5′)], 4.50 and 4.70 [1H, 2xm, CH(2′)],5.40 {1H, br m, CH(4′)], 6.91 and 6.98 (1H, s, CHPh₂), 7.15-7.32 (10H,m, phenyl CH), 7.46-7.62 (3H, m, benzoyl m- and p-CH), 7.70-7.94 [3H, m,benzoyl o-CH and CH(5)], 8.37 [1H, d, J=5.7 Hz, CH(6)], 8.67 (1H, br s,NH); δ_(C) (50.28 MHz; CDCl₃) 27.9 and 28.3 (Boc CH₃ rotamers), 35.1 and36.0 [CH₂(3′) rotamers], 51.7 and 52.1 [CH₂(5′) rotamers], 57.6 and 57.9[CH(2′) rotamers], 74.1 and 75.2 [CH(4′) rotamers], 77.3 and 77.5 (CHPh₂rotamers), 80.2 and 80.4 (Boc C rotamers), 104.7 [CH(5)], 127.1-129.2(aromatic CH), 133.1 and 133.4 (benzoyl C), 140.0-140.3 (aromatic C),154.0 and 154.4 (Boc CO rotamers), 159.6 [C(2)], 160.5 [CH(6) rotamers],163.8 [C(4)], 166.3 (benzoyl CO), 170.9 and 171.1 (ester CO); m/z (ESMS) 595 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1738s (C═O), 1687s (C═O).

[0093] N-tert-Butoxycarbonyl-cis-4-(N₂-isobutyrylguanin-9-yl)-D-prolinediphenylmethyl ester (14)

[0094] To a stirred suspension of the trans-D alcohol (2c) (0.400 g,1.00 mmol), N₂-isobutyryl-O₆-nitrophenylethylguanine (0.360 g, 1.00mmol) and triphenylphosphine (0.294 g, 1.10 mmol) in anhydrous dioxane(10 ml) at room temperature was slowly added DEAD (182 μL, 1.10 mmol)under argon. Another two aliquots of DEAD (91 ml, 0.55 mmol each) wereadded during a period of 36 h. The resulting clear yellow solution wasevaporated and the residue chromatographed (SiO₂, ethyl acetate, R_(f)0.46) to give the O₆-nitrophenylethyl derivative as a white foam (0.634g, contaminated with diethyl hydrazinedicarboxylate). This was dissolvedin dry pyridine (5 ml) containing DBU (300 μL, 2.00 mmol) and thesolution stirred at room temperature overnight under argon. The reactionmixture was diluted with dichloromethane and washed with 5% HCl andwater and then evaporated to dryness. The residue was purified by columnchromatography (SiO₂, ethyl acetate-methanol 20:1) to give the productas a white foam (0.258 g, 43% from 2c). Recrystallisation from ethylacetate-petroleum ether (b.p. 40-60° C.) gaveN-tert-butoxycarbonyl-cis-4-(N₂-isobutyrylguanin-9-yl)-D-prolinediphenylmethyl ester (I4) as a white crystalline solid m.p. 140-145° C.,(Found C, 64.0; H, 5.7; N, 13.6%; C₃₂H₃₆N₆O₆ requires C, 64.0; H, 6.0;N, 14.0%), δ_(H) (200 MHz; CDCl₃) 1.18-1.41 [15H, m, Boc and (CH ₃)₂CH],2.30 (1H, br m) and 2.75-2.95 (2H, br m) [CH ₂(3′) and (CH₃)₂CH], 3.78and 4.05 [2H, br m, CH ₂(5′)], 4.45-4.63 [1H, 2xm, CH(2′)], 4.90 [1H, brm, CH(4′)], 6.77 and 6.80 (1H, s, CHPh₂), 7.15-7.30 (10H, m, phenyl CH),7.60 and 7.66 [1H, 2xs, CH(8)]; δ_(C) (50.28 MHz; CDCl₃) 18.9[(CH₃)₂CH], 27.9 and 28.2 (Boc CH₃ rotamers), 35.8 [CH₂(3′)], 50.2 and50.7 [CH₂(5′) rotamers], 51.9 and 52.3 [CH(4′) rotamers], 57.6 [CH(2′)],60.4 [(CH₃)₂ CH], 77.7 and 77.9 (CHPh₂ rotamers), 81.1 (Boc C), 121.1[C(5)], 127.0-128.7 (aromatic CH), 137.2 [CH(8)], 139.5 (aromatic C),148.2 and 149.1 [C(2)/C(6)], 153.6 and 154.2 (Boc CO rotamers), 156.1[C(4), 171.0 (ester CO), 180.5 (amide CO); m/z (ES MS) 601 (M+H⁺, 100%);λ_(max) (CHCl₃)/nm 255sh (ε/dm³.mol⁻¹.cm⁻¹ 1.5×10⁴), 282 (1.2×10⁴);[α]^(D) ²³ +37.8 (c=0.545, CHCl₃).

[0095] Procedure for Selective Deprotection of N-Boc Group inDiphenylmethyl Esters (3a, 3b and 3c) and Synthesis of N-Fmoc DipeptideDiphenylmethyl Esters (4a, 4b and 4c)

[0096] The Boc-protected monomer (3a, 3b, 3c) was dissolved in THF (ca.10 ml/mmol), saturated methanolic HCl (ca. 10 ml/mmol) was added and thesolution was stirred at room temperature for 3-12 h. The solvents wereremoved under reduced pressure. The residue was taken up in dry dioxaneand DIEA (ca. 2 eq excess) was added until the solution was slightlybasic (pH 8) when applied to a piece of moist pH paper. Fmoc-glycinepentafluorophenyl ester (1 eq. excess) was then added and the solutionstirred at room temperature overnight. The reaction mixture wasevaporated to dryness and the residue purified by column chromatography(SiO₂, dichloromethane:acetone 10:1).

[0097]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₃-benzoylthymin-1-yl)-L-prolinediphenylmethyl ester (4a) was obtained as a white solid (99%, startingfrom 5.8 mmol of 3a) after column chromatography. Recrystallisation fromethanol gave fine colourless needles, m.p. 201-204° C., (Found C, 71.5;H, 5.0; N, 7.0%; C₄₇H₄₀N₂O₈ requires C, 71.6; H, 5.1; N, 7.0%), δ_(H)(200 MHz; CDCl₃) 1.72 and 1.85 (3H, 2xs, thymine CH ₃ rotamers), 2.10,2.45, 2.72 and 2.95 [2H, 4xm, CH ₂(3′) rotamers], 3.60-3.82 and3.95-4.09 [4H, br m, CH ₂(5′) and Gly CH ₂], 4.25 (1H, t, J=7.1Hz, Fmocaliphatic CH), 4.40 (2H, d, J=7.1 Hz, Fmoc CH ₂), 4.77 [1H, br m, CH(2′)rotamers], 5.15 and 5.35 [1H, 2xm, CH(4′) rotamers], 5.60 and 5.78 [1H,2xbr m, Glycine NH rotamers], 6.86 and 6.93 (1H, 2xs, CHPh₂ rotamers),6.96 and 7.11 [1H, 2xs, CH(6) rotamers], 7.25-7.95 (m, phenyl, Fmoc andbenzoyl aromatic CH); δ_(C) (50.28 MHz; CDCl₃) 12.1 (thymine CH₃), 32.5[CH₂(3′) rotamers, 43.5 (Gly CH₂), 47.0 (Fmoc aliphatic CH), 48.5[CH₂(5′)], 53.2 [CH(4′), 57.8 [CH₂(2′)], 67.3 (Fmoc CH ₂), 78.7 (CHPh₂),112.3 [C(5)], 120.1 (Fmoc aromatic CH)], 125.5-131.8 (aromatic CH),135.8 [CH(6)], 139.4 and 139.6 (phenyl C rotamers), 141.7 and 144.0(Fmoc aromatic C), 150.0 [C(2)], 156.2 (Fmoc CO), 162.4 [C(4)], 167.8(benzoyl CO), 168.9 (peptide CO), 170.8 (ester CO); m/z (FAB+) 811(M+Na⁺, 21%), 789 (M+H⁺, 5), 179 {[(C₆H₄)₂C═CH₂+H]⁺, 23}, 167 (Ph₂CH⁺,100), 105 (PhCO⁺, 22); ν_(max) (KBr)/cm⁻¹ 1751s, 1737s, 1697 and 1657s(C═O); λ_(max) (CHCl₃)/nm 260 (ε/dm³.mol⁻¹.cm⁻¹, 3.1×10⁴); [α]_(D) ²²−41.2 (c=0.50, CHCl₃).

[0098]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-trans-4-(N₃-benzoylthymin-1-yl)-D-prolinediphenylmethyl ester (4b) was obtained as a white solid (85%, startingfrom 5.3 mmol of 3b) after column chromatography. Recrystallisation fromethanol-water gave a white solid, m.p. 125-128° C., (Found C, 71.4; H,5.0; N, 6.6%; C₄₇H₄₀N₄O₈ requires C, 71.6; H, 5.1; N, 7.1%), δ_(H) (200MHz; CDCl₃) 1.88 and 1.94 (3H, 2s, thymine CH ₃ rotamers), 2.30 and 2.60[2H, br m, CH ₂(3′) rotamers], 3.70-4.10 [4H, br m, CH ₂(5′) and Gly CH₂], 4.22 (1H, t, J=7.1 Hz, Fmoc aliphatic CH), 4.39 (2H, d, J=7.0 Hz,Fmoc CH ₂), 4.88 [1H, br m, CH(2′)], 5.13 [1H, br m, CH(4′)], 5.90 and5.98 (1H, 2xbr m, Gly NH), 6.91 and 6.94 (1H, 2xs, CHPh₂ rotamers), 7.16[1H, s, CH(6)], 7.29-8.00 (m, phenyl, benzoyl and Fmoc aromatic CH);δ_(C) (50.28 MHz; CDCl₃) 12.4 (thymine CH₃), 32.0 [CH₂(3′) rotamers],43.2 and 43.4 (Gly CH₂ rotamers), 47.0 (Fmoc aliphatic CH), 48.3[CH₂(5′)], 55.2 and 55.3 [CH(4′) rotamers], 57.9 [CH₂(2′)], 67.3 (FmocCH ₂), 78.6 and 79.3 (CHPh₂ rotamers), 112.0 [C(5)], 120.2 (Fmocaromatic CH), 125.4-131.6 (aromatic CH), 135.6 [CH(6)], 137.2 and 139.2(phenyl C rotamers), 141.5 and 144.1 (Fmoc aromatic C), 150.0 [C(2)],156.9 (Fmoc CO), 162.9 [C(4)], 168.4 (benzoyl CO), 169.4 (peptide CO),170.1 and 170.5 (ester CO); m/z (ES MS) 806 (M+NH₄ ⁺, 28%), 789 (M+H⁺,100); ν_(max) (KBr)/cm⁻¹ 1749, 1702 and 1660 (C═O).

[0099]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₃-benzoylthymin-1-yl)-D-prolinediphenylmethyl ester (4c) was obtained as a white solid (98%, startingfrom 5.7 mmol of 3c) after column chromatography (R_(f) 0.30).Recrystallisation from ethanol-water gave colourless needles m.p.201-203° C. (Found C, 72.4; H, 4.9; N, 7.2%; C₄₇H₄₀N₄O₈ requires C,71.6; H, 5.1; N, 7.1%), δ_(H) (200 MHz; CDCl₃) 1.71 and 1.83 (3H, 2xs,thymine CH ₃ rotamers), 2.06, 2.42, 2.78 and 2.92 [2H, m, CH ₂(3′)rotamers], 3.60-3.82 and 3.904.10 [4H, br m, CH ₂(5′) and Gly CH ₂],4.23 (1H, t, J=7.0 Hz, Fmoc aliphatic CH), 4.39 (2H, d, J=7.1 Hz, FmocCH ₂), 4.73 [1H, br m, CH(2′) rotamers], 5.14 and 5.42 [1H, 2xm, CH(4′)rotamers], 5.60 and 5.68 [1H, 2xbr m, Gly NH rotamers], 6.87 and 6.91(1H, 2xs, CHPh₂ rotamers), 6.96 and 7.09 [1H, 2xs, CH(6) rotamers],7.21-7.92 (m, phenyl, Fmoc and benzoyl aromatic CH); m/z (ES MS) 806(M+NH₄ ⁺, 98%), 789 (M+H⁺, 100); ν_(max) (KBr)/cm⁻¹ 1751, 1737, 1697 and1657 (C═O); λ_(max) (CHCl₃)/nm 260 (c/dm³mol⁻¹.cm⁻¹, 3.4×10⁴); [α]_(D)²² +41.5 (c=0.50, CHCl₃).

[0100] Procedure for Selective Deprotection of N-Boc Group inDiphenylmethyl Esters (8, 11 and 14) and Synthesis of N-Fmoc DipeptideDiphenylmethyl Esters (9, 12and 15)

[0101] The Boc-protected monomer (8,11,14) and p-toluenesulfonic acidmonohydrate (5 eq.) was dissolved in acetonitrile (ca. 5 ml/mmol) andthe resulting solution was stirred at room temperature overnight. Thesolvent was removed under reduced pressure and the residue was dissolvedin DMF (ca. 5-10 ml/mmol). DIEA (5 eq. excess) was added until thesolution was slightly basic (pH 8) when applied to a piece of moist pHpaper followed by HOBt.H₂O (1.2 eq.) and Fmoc-glycine pentafluorophenylester (1.2 eq.) and the reaction mixture stirred at room temperatureovernight. The reaction mixture was diluted with dichloromethane andwashed with saturated aqueous NaHCO₃ and water. Evaporation gave thecrude product which was purified by column chromatography.

[0102]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₆benzoyladenin-9-yl)-D-profinediphenylmethyl ester (9) was obtained as a white foam (85%, startingfrom 1.28 mmol of 8) after column chromatography (SiO₂, 10% methanol inethyl acetate), m.p. 130-133° C., (Found C, 71.0; H, 4.8; N, 12.2%;C₄₇H₃₉N₇O₆ requires C, 70.8; H, 4.9; N, 12.3%), 8H (200 MHz; CDCl₃) 2.58and 2.83 [2H, 2xm, CH ₂(3′)], 3.78-4.40 [m, unresolved CH ₂(5′), Gly CH₂, Fmoc aliphatic CH and CH ₂], 4.78 [1H, m, CH(2′)], 5.05 and 5.28 1H,2xm, CH(4′) rotamers], 6.13 (1H, brt, Gly NH), 6.76 (1H, 2xs, CHPh₂rotamers), 7.20-7.75 (m, benzoyl m- and p-CH, phenyl and Fmoc aromaticCH), 7.96-8.00 (2H, d, J=7.1 Hz, benzoyl o-CH), 8.23 [1H, s, CH(8)],8.70 (1H, s, CH(2)], 9.48 (1H, br s, benzamide NH); δ_(C) (50.28 MHz;CDCl₃) 33.5 [CH₂(3′)], 43.3 (Gly CH₂), 47.0 (Fmoc aliphatic CH), 49.4[CH₂(5′)], 53.2[CH(4′)], 57.8 [CH(2′)], 67.1 (Fmoc CH₂), 78.6 (CHPh₂),120.1 (Fmoc aromatic CH), 123.5(C(5)], 125.2-128.9 and 133.0 (aromaticCH), 133.8, 139.4 and 141.4 (aromatic C), 142.0 [CH(8)], 144.1 (aromaticC), 150.0 [C(4)], 152.1 [C(6)], 152.7 [CH(2)], 156.9 (Fmoc CO), 165.4(benzoyl CO), 168.6 (Gly CO), 170.4 (ester CO); m/z (ES MS) 798 (M+H⁺,100%); ν_(max) (KBr)/cm⁻¹ 1718 (C═O), 1668 (C═O); [α]_(D) ²² +18.6(c=0.21, CHCl₃).

[0103]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₄-benzoylcytosin-1-yl)-D-prolinediphenylmethyl ester (12) was obtained as a white foam (70%, startingfrom 1.12 mmol of 11) after column chromatography (SiO₂, ethylacetate-methanol 20:1). Recrystallisation from ethanol gave a whitesolid, m.p. 131-133° C., (Found C, 71.2; H, 4.9; N. 9.0%; C₄₆H₃₉N₅O₇requires C, 71.4; H, 5.1; N, 9.0%), δ_(H) (200 MHz; CDCl₃) 2.20 and 2.85[2H, 2xm, CH₂(3′)], 3.61-4.40 [m, unresolved CH ₂(5′), Gly CH ₂, Fmocaliphatic CH and CH ₂], 4.76-4.81 [1H, m, CH(2′) rotamers], 5.22 and5.41 (1H, 2xm, CH(4′) rotamers], 6.03 and 6.12 (1H, 2xbr t, Gly NHrotamers), 6.78 (1H, br s, CHPh₂), 7.05-8.00 [m, CH(5), CH(6), benzoyl,phenyl and Fmoc aromatic CH], 9.32 (1H, br s, benzamide NH); δ_(C)(50.28 MHz; CDCl₃) 33.2 [CH₂(3′)], 43.5 (Gly CH₂), 47.0 (Fmoc aliphaticCH), 49.2 [CH₂(5′)], 55.5 [CH(4′)], 57.8 [CH(2′)], 67.1 (Fmoc CH₂), 78.6(CHPh₂), 97.3 [CH(5)], 120.1 (Fmoc aromatic CH), 125.4-129.1 and 133.1(aromatic CH), 133.3, 139.4 and 141.1 (aromatic C), 144.1 (aromatic C),145.6 [CH(6)], 155.8 [C(2)], 156.7 (Fmoc CO), 162.6 and 163.0 [C(4)rotamers], 167.1 (benzoyl CO), 168.6 (Gly CO), 170.6 (ester CO); m/z (ESMS) 774 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1750-1665br (C═O); [α]_(D) ₂₂+20.9 (c=0.21, CHCl₃).

[0104]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₂-isobutyrylguanin-9-yl)-D-prolinediphenylmethyl ester (15) was obtained as a white solid (52%, startingfrom 0.73 mmol of 14) after column chromatography (SiO_(2,) 10% methanolin ethyl acetate). Recrystallisation from ethyl acetate-petroleum ether(b.p. 40-60° C.) gave a white crystalline solid, m.p. 145-150° C.,(Found C, 68.0; H. 5.2; N, 12.0%; C₄₄H₄₁N₇O₇ requires C, 67.8; H, 5.3;N, 12.6%), δ_(H) (200 MHz; CDCl₃) 1.18 and 1.21 [6H, d, J=6.7 Hz, (CH₃)₂CH], 2.31 (1H, br m) and 2.61-2.79 (2H, br m) [CH ₂(3′) and(CH₃)₂CH], 3.89-4.20 [m, unresolved CH ₂(5′), Gly CH ₂ and Fmocaliphatic CH], 4.37 (2H, d, J=6.7 Hz, Fmoc CH ₂), 4.63 [1H, m, CH(2′)],4.82 [1H, m, CH(4′)], 6.10 (1H, br m, Gly NH), 6.77 (1H, s, CHPh₂),7.12-7.36 (m, phenyl and Fmoc aromatic CH), 7.49-7.57 [3H, m, Fmocaromatic CH and CH(8)], 7.69-7.73 (2H, d, J=7.4 Hz, Fmoc aromatic CH),9.83 (1H, br s, isobutyramide NH); δ_(C) (50.28 MHz; CDCl₃) 18.9[(CH₃)₂CH], 35.7 [CH₂(3′)], 42.9 (Gly CH₂), 46.5 (Fmoc CH), 49.0[CH₂(5′)], 52.9 [CH(4′)], 57.7 [CH(2′)], 66.9 (Fmoc CH₂), 78.3 (CHPh₂),120.1 (Fmoc aromatic CH), 120.9 [C(5), 125.2-128.8 (Fmoc aromatic CH),137.5 [CH(8)], 139.4 and 139.6 (aromatic C), 144.0 (aromatic C), 148.3and 148.8 [C(2)/C(6)], 155.8 [C(4)], 157.2 (Fmoc CO), 169.0 (Gly CO),170.2 (ester CO), 180.6 (isobutyramide CO); m/z (ES MS) 780 (M+H⁺,100%); λ_(max) (CHCl₃)/nm 270sh (ε/dm³.mol⁻¹.cm⁻¹, 11.9×10⁴); [α]_(D) ²³+36.7 (c=0.645, CHCl₃).

[0105] N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-4-(thymin-1-yl)prolines(5a), (5b) and (5c)

[0106] The protected dipeptide (4a, 4b or 4c) was treated with 10% HBrin acetic acid (5-10 ml/mmol) at room temperature for 1 h. The volatileswere evaporated under reduced pressure, the residue was triturated withdiethyl ether and then washed with methanol-diethyl ether.

[0107]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(thymin-1-yl)-L-proline(5a) was obtained as a white solid (50%, starting from 2.5 mmol of 4a).Recrystallisation from ethanol-water gave a white solid, m.p.>200° C.,(Found C, 62.5; H, 5.1; N, 10.6%; C₂₇H₂₆N₄O₇ requires C, 62.5; H, 5.1;N, 10.8%), δ_(H) (200 MHz; DMSO-d₆) 1.75 (3H, br s, thymine CH ₃),2.00-2.35 [2H, br m, CH ₂(3′) rotamers], 3.30-4.00 [br m, CH ₂(5′) andGly CH ₂ obscured by the water signal], 4.15-4.30 (3H, br m, Fmocaliphatic CH and CH ₂), 4.50-4.65 [1H, br m, CH(2′) rotamers], 4.80-4.85and 4.95-5.05 [1H, br m, CH(4′) rotamers], 7.25-7.45 (4H, m, Fmocaromatic CH), 7.52 [1H, br s, CH(6) rotamers], 7.70 and 7.85 (4H, 2xd,J=7.1 Hz, Fmoc aromatic CH); m/z (FAB) 541 (M+Na⁺, 9%), 179{[(C₆H₄)₂C═CH₂.H]⁺, 81}, 165 (32), 119 (30), 103 (44), 85 (83), 77(32),59 (85), 47(100); ν_(max) (KBr)/cm⁻¹ 1731 (C═O), 1703s (C═O), 1678(C═O); [α]_(D) ²³ −4.13 (c=0.63, DMF).

[0108]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-trans-4-(thymin-1-yl)-D-proline(5b) was obtained as a white solid (57%, starting from 5.3 mmol of 4b)m.p.>200° C., δ_(H) (200 MHz; DMSO-d₆) 1.70 (3H, br s, thymine CH ₃),2.05-2.15 and 2.40-2.60 [br m, CH ₂(3′) rotamers, obscured by the DMSOsignal], 3.50-4.00 [br m, CH ₂(5′) and Gly CH ₂], 4.15-4.30 (3H, br m,Fmoc aliphatic CH and CH ₂), 4.35-4.45 and 4.75-4.85 [1H, br m, CH(2′)rotamers], 4.90-5.00 and 5.05-5.10 11H, m, CH(4′) rotamers], 7.25-7.45(4H, m, Fmoc aromatic CH), 7.55 [1H, br s, CH(6)], 7.68 (4H, 2xd, J=7.1Hz, Fmoc aromatic CH); m/z (FAB) 519 (M+H⁺, 6%), 179 {[(C₆H₄)₂C═CH₂.H]⁺,34}, 85 (100), 59 (23), 47 (32).

[0109]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(thymin-1-yl)-D-proline(5c) was obtained as a white solid (42%, starting from 6.8 mmol of 4c),m.p.>200° C., δ_(H) (200 MHz; DMSO-d₆) 1.75 (3H, br s, thymine CH ₃),2.11 and 2.52 [2H, 2xbr m, CH ₂(3') rotamers], 3.50-4.00 [br m, CH ₂(5′)and Gly CH ₂], 4.18-4.30 [4H, br m, CH(2′) and Fmoc aliphatic CH and CH₂], 4.73 and 4.98 [1H, 2xbr m, CH(4′) rotamers], 7.28-7.41 (4H, m, Fmocaromatic CH), 7.51 (1H, m, Gly NH), 7.54 [1H, br s, CH(6) rotamers],7.72 and 7.88 (4H, 2xd, J=7.1 Hz, Fmoc aromatic CH); m/z (FAB) 541(M+Na⁺, 2%), 519 (M+H⁺, 5), 179 (33), 103 (17), 85 (100), 77(18), 59(43), 47(45); [α]_(D) ²³ +4.26 (c=0.61, DMF).

[0110] N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-4-(thymin-1-yl)prolinePentafluorophenyl Esters (6a), (6b) and (6c)

[0111] A suspension of the Fmoc-dipeptide (5a, 5b or 5c) (1.0 mmol),pentafluorophenol (1.1 mmol) and dicyclohexylcarbodiimide (1.1 mmol) indichloromethane (5 ml) was stirred at room temperature for 2-3 h. Theprecipitated dicyclohexylurea was filtered off and washed withdichloromethane. Evaporation of the filtrate followed by columnchromatography (SiO₂, ethyl acetate) gave the product as white foamwhich in most cases could be made crystalline by trituration withether-petroleum ether, filtered and air dried.

[0112]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(thymin-1-yl)-L-prolinepentafluorophenyl ester (6a) was obtained from (5a) as a white solid(0.574 g, 84%), m.p. 124-126° C., δ_(H) (200 MHz; CDCl₃) 1.94 (3H, s,thymine CH ₃), 2.26-2.42 and 2.85-3.00 [2H, m, CH ₂(3′)], 3.66-3.75 and3.95-4.26 [m, unresolved CH ₂(5′), Gly CH ₂, Fmoc aliphatic CH],4.38-4.42 (2H, d, J=7.1 Hz, Fmoc CH ₂), 4.85-4.93 [1H, m, CH(2′)],5.34-5.42 [1H, m, CH(4′)], 5.80-5.82 (1H, brt, Gly NH), 7.10 [1H, s,CH(6)], 7.31-7.44, 7.60-7.63 and 7.75-7.79 (8H, m, Fmoc aromatic CH),9.50 (1H, s, thymine NH); δ_(F) (235.35 MHz; CDCl₃) −162.0 (dd, J=18.1,21.4 Hz) and −161.2 (t, J=19.6 Hz) (m-F major and minor rotamers),−157.0 (t, J=21.8 Hz) and -156.2 (t, J=21.7 Hz) (p-F major and minorrotamers), −153.1 (d, J=18.5 Hz) and −152.8 (d, J=17.7 Hz) (o-F minorand major rotamers). The ratio of major:minor rotamers was ca. 15:1; m/z(ES MS) 685.1 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1801 (C═O), 1675br (C═O);[α]_(D) ²³ −15.9 (c=0.630, CHCl₃);

[0113]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-trans-4-(thymin-1-yl)-D-prolinepentafluorophenyl ester (6b) was obtained from (5b) as a white solid(0.400 g, 58%), m.p. 115-124° C., δ_(H) (200 MHz; CDCl₃) 1.94 (3H, s,thymine CH ₃), 2.49-2.61 and 2.79-2.95 [2H, m, CH ₂(3′)], 3.79-4.13 [4H,m, CH ₂(5′) and Gly CH _(2], 4.22) (1H, t, J=6.7 Hz, Fmoc aliphatic CH)4.37-4.41 (2H, d, J=7.1 Hz, Fmoc CH ₂), 5.04-5.18 [2H, m, CH(2′) andCH(4′)], 5.78-5.82 (1H, brt, Gly NH), 7.00 and 7.02 [1H, 2xs, CH(6)rotamers], 7.27-7.44, 7.58-7.61 and 7.74-7.78 (8H, m, Fmoc aromatic CH),9.17 and 9.21 (1H, 2xs, thymine NH rotamers); δ_(F) (235.35 MHz;CDCl₃)-162.1 (t, J=19.3 Hz) and −161.4 (t, J=21.8 Hz) (m-F major andminor rotamers), −157.2 (t, J=22.5 Hz) and −156.3 (t, J=19.4 Hz) (p-Fmajor and minor rotamers), −153.3 (d, J=19.5 Hz) and −153.0 (d, J=20.0Hz) (o-F minor and major rotamers); ν_(max) (KBr)/cm⁻¹ 1797 (C═O),1679br (C═O); [α]_(D) ²³ +30.0 (c=0.73, CHCl₃).

[0114]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(thymin-1-yl)-D-prolinepentafluorophenyl ester (6c) was obtained from (5c) as a white solid(0.520 g, 76%), m.p. 122-126° C., δ_(H) (200 MHz; CDCl₃) 1.94 (3H, s,thymine CH ₃), 2.26-2.42 and 2.85-3.00 [2H, m, CH ₂(3′)], 3.66-3.75 and3.95-4.26 [5H, m, CH ₂(5′), Gly CH ₂ and Fmoc aliphatic CH], 4.384.42(2H, d, J=7.1 Hz, Fmoc CH ₂), 4.85-4.93 [1H, m, CH(2′)], 5.34-5.42 [1H,m, CH(4′)], 5.80-5.82 (1H, brt, Gly NH), 7.10 [1H, s, CH(6)], 7.31-7.44,7.60-7.63 and 7.75-7.79 (8H, m, Fmoc aromatic CH), 9.50 (1H, s, thymineNH); δ_(F) (235.35 MHz; CDCl₃) −162.0 (dd, J=18.1, 21.4 Hz) and −161.2(t, J=19.6 Hz) (m-F major and minor rotamers), −157.0 (t, J=21.8 Hz) and−156.2 (t, J=21.7 Hz) (p-F major and minor rotamers), −153.1 (d, J=18.5Hz) and −152.8 (d, J=17.7 Hz) (o-F minor and major rotamers). The ratioof major:minor rotamers was ca. 15:1; ν_(max) (KBr)/cm⁻¹ 1800 (C═O),1683br (C═O); [α]_(D) ²³ +16.3 (c=0.645, CHCl₃).

[0115] Procedure for Deprotection of Diphenylmethyl Esters and Synthesisof Fmoc-dipeptide Pentaflurophenyl Esters (10, 13 and 16)

[0116] The Fmoc dipeptide diphenylmethyl ester (9, 12 or 15) was treatedwith trifluoroacetic acid (ca. 5-10 ml/mmol) containing anisole (50μl/ml TFA) for 2-3 h. The volatiles were evaporated under reducedpressure and the residue was triturated and washed with diethyl ether.The free acid was obtained as a white solid in nearly quantitative yieldafter drying over NaOH pellets in vacuo. This was dissolved in 1:1DMF:dichloromethane (5 ml/mmol) and pentafluorophenol (1.5 eq.) and DCCI(1.5 eq.) was added with stirring at room temperature. The reactionmixture was stirred at room temperature for 1-3 h (tlc). The DCUprecipitate was filtered off and washed with dichloromethane. Thecombined organic phase was washed with water, dried (MgSO₄) andevaporated under reduced pressure. The residue was triturated withdiethyl ether:petroteum ether 40-60° C. or re-precipitated from suitablesolvents to give the product which contained a trace of DCU (˜10%) asthe only impurity according to ¹H nmr but was pure enough for solidphase synthesis.

[0117]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₆-benzoyladenin-9-yl)-D-prolinepentafluorophenyl ester (10) was obtained as a white solid (83%,starting from 0.5 mmol of 9), m.p. 125-130° C., 5H (200 MHz; CDCl₃)2.86-3.02 and 3.10-3.24 [2H, m, CH ₂(3′)], 3.98-4.42 [7H, m, CH ₂(5′),Gly CH ₂ and Fmoc aliphatic CH and CH ₂], 4.97-5.06 [1H, t, J=8.5 Hz,CH(2′)], 5.31-5.42 [1H, m, CH(4′)], 5.70-5.74 (1H, br t, J=3.8 Hz, GlyNH), 7.27-7.79 (11H, m, Fmoc aromatic CH and benzoyl m- and p-CH),8.02-8.06 (2H, d, J=6.7 Hz, benzoyl o-CH), 8.13 [1H, s, CH(8)], 8.80[1H, s, CH(2)], 9.00 (1H, br s, benzamide NH); m/z (ES MS) 798 (M+H⁺,100%); λ_(max) (KBr)/cm⁻¹ 1798 (C═O), 1671 br (C═O).

[0118]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₄-benzoylcytosin-1-yl)-D-prolinepentafluorophenyl ester (13) was obtained as a white solid (81%,starting from 0.17 mmol of 12), m.p. 133-137° C., δ_(H) (200 MHz; CDCl₃)2.46-2.60 and 290-3.12 [2H, m, CH ₂(3′)], 3.80-4.43 [7H, m, CH ₂(5′),Gly CH ₂ and Fmoc aliphatic CH and CH ₂], 4.93-5.01 [1H, t, J=7.7 Hz,CH(2′)], 5.41-5.49 [1H, m, CH(4′)], 5.72-5.78 (1H, br t, J=4.5 Hz, GlyNH), 7.27-7.79 [m, CH(6), CH(5), Fmoc aromatic CH and benzoyl CH], 7.90(1H, br s, benzamide NH); δ_(F) (235.35 MHz; CDCl₃) −162.0 (t, J=21.1Hz)and −161.3 (t, J=19.1 Hz) (m-F major and minor rotamers), −157.2 (t,J=19.4 Hz) and −156.4 (t) (p-F major and minor rotamers), −152.6 (d,J=20.0 Hz) (o-F); m/z (ES MS) 774 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1797(C═O), 1669 (C═O).

[0119]N-(N-Fluoren-9-ylmethoxycarbonylglycyl)-cis-4-(N₂-isobutyrylguanin-9-yl)-D-prolinepentafluorophenyl ester (16) was obtained as a white solid (63%,starting from 0.16 mmol of 15), m.p. 146-150° C., 6H (200 MHz; CDCl₃)1.22-1.27 16H, 2xd, J=6.9 Hz, (CH ₃)₂CH], 2.60-2.82 and 2.97-3.12 [3H,m, CH ₂(3′) and (CH₃)₂CH], 4.08-4.30 [5H, m, CH ₂(5′), Gly CH ₂ and Fmocaliphatic CH], 4.37-4.41 (2H, d, J=7.2 Hz, Fmoc CH ₂), 4.85-4.94 [1H. t,J=8.3 Hz, CH(2′)], 4.99-5.06 [1H, m, CH(4′)], 5.79-5.84 (1H, br t, J=4.5Hz, Gly NH), 7.27-7.44 (4H, m, Fmoc aromatic CH), 7.56-7.60 (2H, d,J=7.4 Hz, Fmoc aromatic CH), 7.67 [1H, s, CH(8)], 7.74-7.78 (2H, d,J=7.4 Hz, Fmoc aromatic CH), 8.95 (1H, s, isobutyramide NH); m/z (ES MS)780 (M+H⁺, 100%); ν_(max) (KBr)/cm⁻¹ 1798 (C═O), 1680br (C═O).

[0120] Novasyn™ TGR resin (−0.23 mmol free NH₂ group/g) (Fmoc/O^(t)Bustrategy), were obtained from Calbiochem-Novabiochem Ltd.

[0121] The protected amino acids and derivatives and the couplingreagents (HBTU, PyBrop) were also obtained from the same source.Trifluoroacetic acid (98%) was obtained from Avocado Research ChemicalsLtd. All other reagents were obtained at highest purity grade availableeither from Aldrich Chemical Company Ltd. or Lancaster Synthesis Ltd.and were used as received. Reagents for the Kaiser test were preparedaccording to the literature.²³

[0122] DMF was peptide synthesis grade obtained from Rathburn ChemicalsLtd. and used without further purification. All other solvents used forthe synthesis and purification were hplc grade solvents obtained fromRathburn. Deionised water was obtained from an Elga Maxima Ultra-Purewater purification system.

[0123] Samples for reverse phase hplc analysis were dissolved in asuitable aqueous solvent and filtered through a teflon filter (0.47[pore size, Anachem Ltd.). Hplc was performed on a Waters 990+ systemwith a diode array detector. A Waters μBondapak C-18 semi-preparativereverse phase hplc column (0.78×30 cm, P/N 84176) was used for bothanalysis and preparative purposes. Peak monitoring and data analysiswere performed on Waters 990 software running on a NEC IBM-PC/ATcompatible computer with 80286180287 microprocessors. The samples wererecovered from hplc fractions by freeze drying on a VirTis Freezemobile5SL freeze drier. Electrospray mass spectra of the peptide nucleic acidswere recorded by on a VG Biotech-BioQ or VG Biotech Plafform massspectrometers.

[0124] Small Scale Solid Phase Synthesis of Peptide Nucleic Acids usingFmoc/O^(t)Bu-fragment Coupling Strategy

[0125] In a polyethylene syringe (1 ml) equipped with a removablestainless steel needle fitted with a glass wool plug at the junction wasplaced the Novasyn TGR resin [preloaded with Fmoc-Lys(Boc)—OH 0.23mmol/g; 10-25 mg, ca. 2.5-5 μlmol]. The needle was then inserted througha rubber septum fitted to a Büchner flask. Washing was done by addingsolvent from the top of the syringe with the plunger removed and suckinginto the receiver flask by a water aspirator. For the deprotection,coupling and capping stages, the plunger was re-attached and the reagentwas taken up through the needle. Deprotection of the Fmoc group wasaccomplished by treatment of the Fmoc-peptide resin with freshlyprepared 20% piperidine in DMF (1.0 ml on a 5 pmol scale synthesis) for20 min with occasional agitation. After the specified period of time thereagent was ejected by depressing the plunger and washing carried out asdescribed above. The solution from the deprotection stage containing thedibenzofulvene-piperidine adduct was collected and the OD₂₆₄ measured toassess the efficiency of the previous coupling stage. The resin had tobe washed exhaustively to ensure complete removal of the piperidine. Thecoupling was carried out similarly using typically 4 equivalents of theFmoc-dipeptide pentafluorophenyl ester and 4 equivalents of HOBt.H₂O inDMF with a final concentration of the pentafluorophenyl ester atapproximately 0.1 M. Generally the coupling was completed within 3 h andno second coupling was required. In cases where incomplete coupling wassuspected, the peptide resin was treated with 5% Ac₂O in DMF (1 ml for a5 μmol scale synthesis) for 30 min at room temperature to prevent theformation of deletion sequences. The acetylating mixture was ejected andthe reaction vessel flushed with DMF 3 times before thedeprotection-coupling-capping steps repeated until the last peptidefragment had been added. The N-terminal Fmoc group was removed by 20%piperidine in DMF and the resin was washed with DMF. If the sequencecontained only is thymine or if the exocyclic amino protecting groupswere to be retained, the cleavage of the peptide nucleic acid from theresin with trifluoroacetic acid was performed directly. However, when afully deprotected peptide nucleic acid containing adenine, cytosineand/or guanine was required, the resin-bound peptide was capped with aBoc group prior to the cleavage as described below.

[0126] Deprotection of Exocyclic Amino Protecting Groups on Nucleobases(A, C and/or G Containing Sequences Only) Via a Temporary Boc-protection

[0127] The resin-bound peptide containing a free amino terminus (5 μmolscale synthesis) was treated with a solution of di-t-butyl dicarbonate(50 μL, 22 μmol) and DIEA (35 μL, 20 μmol) in DMF (150 μL) at roomtemperature. Kaiser testing indicated complete reaction after 3 h. Theresin was washed several times with DMF and then the exocyclic aminoprotecting groups were removed by treatment with 1:1 mixture ofethylenediamine and 95% ethanol (200 μL) at room temperature overnight.For cytosine containing sequences, the peptide nucleic acids weretreated with 1:1 concentrated aqueous ammonia-dioxane at 55° C. for 16 hinstead of ethylenediamine-ethanol to avoid the transamination reactionat cytosine residues. The resin-bound deprotected peptide nucleic acidfrom either method was washed with DMF then methanol and air dried.

[0128] Hybridisation Studies with Peptide Nucleic Acids

[0129] Sterile deionised water was used for all experiments involvingoligonucleotides and nucleopeptides. Poly(2′-deoxyadenylic acid)[poly(dA)] (sodium salt, average Mr 8.9×104) was obtained from PharmaciaBiotech. Polyadenylic acid [poly(rA)] (potassium salt, average Mr 7×106)was obtained from Fluka Chemicals Ltd. Oligonucleotides were synthesisedby the phosphoramidite method on Applied Biosystems DNA synthesisers,model 380B or model 394. The exocyclic amino protecting groups wereremoved by heating with concentrated aqueous ammonia solution at 55° C.overnight and the solvent was evaporated under vacuum at 40” C on aSavant SpeedVac vacuum concentrator (Savant Instruments). Theoligonucleotides were purified by ethanol precipitation in the presenceof ammonium acetate, reverse phase hplc (0.1 M triethylammonium acetatebuffer-acetonitrile gradient system) or by an OligonucleotidePurification Cartridge (OPC column, Applied Biosystems Inc.) asappropriate and were stored as a concentrated aqueous solution atneutral pH at −20” C.

[0130] The concentration of oligonucleotide, nucleic acid andnucleopeptide solutions was determined from the absorbance at 260 nm(OD260). The following molar extinction coefficients (e) were usedwithout compensation for the hypochromic effect due to the formation ofordered secondary structure of single-stranded nucleic acids: A, 15.4ml/mmol.cm; T, 8.8 ml/mmol.cm. The same values were also used fornucleopeptides.

[0131] Temperature Dependent UV Measurements

[0132] All the Tm measurements were carried out on a Varian CARY 13 UVspectrophotometer equipped with a temperature control system. Themachine was controlled by a CARY 13 software running on an IBM PS/2system model 30/286. The sample for Tm measurement was prepared bymixing calculated amounts of stock oligonucleotide and nucleopeptidesolutions together and the calculated amounts of NaCl and sodiumphosphate buffer (pH 7.0) were then added as stock solutions and thefinal volumes were adjusted to 3.0 ml by addition of water. The sampleswere transferred to a 10 mm quartz cell with teflon stopper andequilibrated at the starting temperature for at least 30 min. The OD260was recorded in steps from 5-95° C. (heater temperature) with atemperature increment of 0.25-0.5° C./min, The results were normalisedby dividing the absorbance at each temperature by the initialabsorbance. Analysis of the data was performed on a KaleidaGraphsoftware 2.1.3 (Abelbeck Software) running on a Macintosh LC IIIcomputer. The OD260 was normalised by dividing with the initial OD260.The melting temperatures were determined from the maxima of the firstderivative plots of the normalised OD260 against temperature. Percenthyperchromicity was calculated from the ratio of the OD260 at the end ofexperiment to the initial OD260.

[0133] UV-titration Experiment

[0134] The UV titration experiment was performed on a Pye Unicam SP8-100UV spectrophotometer at room temperature. To a solution containing thecis-D decathymine peptide nucleic acid (OD260=0.145; 16.5 mM dTnucleotide) and 10 mM sodium phosphate buffer pH 7.0 (2.0 ml) was addeda 10 ml aliquot of a concentrated stock solution of poly(rA)(OD260=4.34; 0.28 mM dA nucleotide) in 10 mM sodium phosphate buffer pH7.0. The absorbance was read against a blank (10 mM sodium phosphate)and more poly(rA) aliquots were added until a total volume of 500 ml hadbeen added. The ratio of the observed OD260 and the calculated OD260(equation 2) were plotted against the mole ratio of T:A nucleotide(equation 3) and the stoichiometry was determined from the inflectionpoint. $\begin{matrix}\begin{matrix}{{{calcd}.\quad {OD260}} = \quad {\left\lbrack {{{{OD260}(T)} \times V_{T}} + {{{OD260}(A)} \times V_{A}}} \right\rbrack/\left\lbrack {V_{T} + V_{A}} \right\rbrack}} \\\left. {= \quad {{\left\lbrack {{0.145 \times 2} + {4.34 \times {V_{A}({ml})}}} \right\rbrack/2} + {V({ml})}}} \right\rbrack\end{matrix} & (2) \\\begin{matrix}{{{ratio}\quad {of}\quad T\text{:}A} = \quad {\left\lbrack {ɛ_{A} \times {{OD260}(T)} \times V_{T}} \right\rbrack/\left\lbrack {ɛ_{T} \times {{OD260}(A)} \times V_{A}} \right\rbrack}} \\{= \quad {\left\lbrack {15.4 \times 0.145 \times 2} \right\rbrack/\left\lbrack {8.8 \times 4.34 \times {V_{A}({ml})}} \right\rbrack}}\end{matrix} & (3)\end{matrix}$

[0135]N-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butyl-L-seryl)-cis-4-(benzoylthymin-1-yl)-D-prolinediphenylmethyl ester

[0136] N-Boc-D-Pro(cis-4-BzT)-ODpm (350 mg, 0.58 mmol) was dissolved inacetonitrile (5 mL). p-Toluenesulfonic acid monohydrate (552 mg, 2.9mmol) was added and the solution was stirred at room temperature for 1.5h, after which tlc indicated complete deprotection of the N-Boc group.Diisopropylethylamine (515 ml, excess) and DMF (5 ml) were added and thereaction stirred under argon. In a separate reaction vessel, a mixtureof Fmoc-L-Ser(O^(t)Bu)—OH (268 mg, 0.70 mmol) HOBt.H₂O (118 mg, 0.77mmol) and DCCI (160 mg, 0.78 mmol) in DMF (2 ml) was stirred at roomtemperature. After 2 hours, a white precipitate of dicyclohexylureaformed which was removed by filtration and the filtrate transferred tothe first reaction vessel. The reaction mixture was stirred at roomtemperature for a further 3 h then diluted with dichloromethane (50 ml)followed by washing with saturated aqueous NaHCO₃ and water. The organicphase was dried (MgSO₄) and the solvent removed under reduced pressureto give the crude product as an oil which was purified by columnchromatography (SiO₂; dichloromethane: acetone 20:1). The product(R_(f)=0.43) was obtained as a white foam (455 mg, 89%), 5H (200 MHz,CDCl₃) 1.15 (9H, s, ^(t)Bu), 1.81 (3H, s, thymine CH₃), 2.01-2.15 and2.41-2.83 [2H 2xm, CH₂(3′)], 3.45-3.54(1H, m) 3.63-3.71 (1H, m)3,80-3.89 (1H, m) [CH₂(5′) and Ser CH _(a)H_(b)], 4.19-4.54(5H, m,unresolved Fmoc aliphatic CH, CH₂ and Ser CH_(a)H_(b)), 4.71-4.79 [2H,m, CH(2′) and Ser CH], 5.18-5.30 [CH(4′)], 5.73-5.77 (1H, d J=8.2 Hz,peptide NH), 6.90 (1H, s, CHPh₂), 7.18 [1H, s, CH(6)], 7.22-7.66 (m,phenyl, Fmoc and benzoyl aromatic CH), 7.90-7.94 and 8.04-8.08 (2×2H,2xd, Fmoc aromatic CH); d_(c) (50.28 MHz; CDCl₃) 12.3 (thymine CH₃),27.3 (^(t)Bu CH₃), 33.1 [CH₂(3′)], 47.0 (Fmoc aliphatic CH), 49.5[CH₂(5′)], 52.5 and 52.9 [Ser C_(a)H and CH(4′)], 57.5 [CH(2′)], 63.3and64.0 (Ser CH₂ rotamers), 67.3 (Fmoc CH₂), 73.9 (^(t)Bu C), 78.5 (CHPh₂),112.0[C(5)], 120.2 (Fmoc aromatic CH), 125.2-131.7 (aromatic CH), 135.4and 136.0[CH(6) and benzoyl p-CH], 139.5 and 139.8 (aromatic Crotamers), 141.5, 143.9 and 144.1 (Fmoc aromatic C), 150.1 [C(2)], 156.2(Fmoc CO), 162.7 [C(4)], 169.1 (benzoyl CO), 170.5 (peptide CO), 170.8(ester CO); M/z (APCI+) 931 (M+C₄H₈ ⁺, 90%) 876 (M+H⁺, 88), 654, 587,550, 503, 409, 165, 105 (PhCO+, 100).

[0137]N-(N-Fluoren-9-ylmethoxycarbonyl-O-t-butyl-D-seryl)-cis-4-(benzoylthymin-1-yl)-D-prolinediphenylmethyl ester was obtained in analogous manner to the L-Ser-D-Prodiastereomer as a white foam [78%, starting from 1.08 mmol ofBoc-protected proline derivative and 1.20 mmol of Fmoc-D-Ser(OtBu)—OH]after column chromatography: δ_(H) (200 MHz; CDCl₃) 1.18 and 1.21 (9H,2xs, tBu rotamers), 1.70 and 1.85 (3H, 2xs, thymine CH₃ rotamers),2.00-2.17 and 2.49-2.88 [2H, 2xm, CH₂(3′)], 3.37-3.3.72 and 3.99-4.56[m, unresolved CH₂(5′), Ser CH₂ and Fmoc aliphatic CH, CH₂], 4.63-4.84[2H, m CH(2′) and Set CH], 5.21-5.40 [CH (4′)], 5.80-5.84 (1H m, peptideNH, 6.96 (1H, m, CHPh₂ rotamers), 7.23 [1H, s, CH (6)], 7.25-7.97 (m,phenyl, Fmoc and benzoyl aromatic CH); d_(c) (50.28 MHz; CDCl₃) 12.3 and12.4 (thymine CH₃ rotamers), 27.2 (^(t)Bu CH₃), 33.2 [CH₂(3′)], 47.1(Fmoc aliphatic CH), 50.2 [CH₂(5′)], 52.2, 53.0 and 53.7 (Ser CH andCH(4′) rotamers], 57.7and 57.9 [CH(2′) rotamers], 63.3 (Ser CH₂rotamers), 67.2 (Fmoc CH₂), 74.0 and 74.1 (Bu C rotamers), 78.4 and 79.6(CHPh₂ rotamers), 111.8 and 111.9 [C(5) rotamers], 120.2 (Fmoc aromaticCH), 125.4-131.7 (aromatic CH), 135.4 and 136.4 [CH(6) and benzoylp-CH], 139.3 and 139.9 (aromatic C rotamers), 141.6, 144. 0 and 144.1(Fmoc aromatic C), 150.0 [C(2)], 156.1 (Fmoc CO), 162.7 [C(4)], 169.2(benzoyl CO), 170.7 (peptide CO), 170.8 (ester CO); m/z (APCI⁺) 930(M+C₄H₈ ⁺, 26%) 876 (M+H⁺, 38), 708, 653, 587, 550, 503, 167 (Ph₂CH⁺,100).

[0138] General Procedure for Deprotection of the Diphenyfinethyl EsterinN-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butylseryl)-cis-4-(benzoylthymin-1-yl)-D-prolineDiphenylmethyl Ester

[0139] The protected dipeptide was treated with a mixture of saturatedHCl in dioxane (ca. 5.1 M) and dichloromethane (2:1 v/v) (ca 5 ml/mmol)at room temperature and the progress of the reaction was monitored bytlc. Complete cleavage of the Dpm ester was observed after 6-12h. Thereaction mixture was then diluted with dichloromethane and washed withhalf-saturated aq. Na₂HPO₄ and water. Evaporation followed bytrituration with ether/light petroleum gave the product as a whitesolid.

[0140]N-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butyl-L-seryl)-cis-4-(benzoylthymin-l-yl)-D-prolinewas obtained as a white solid [93%, starting from 0.66 mmol ofFmoc-L-Ser(O^(t)Bu)-D-Pro(cis-4-BzT)-ODpm]. ¹H nmr revealed the presenceof impurities, however, the crude product was used for the next stepwithout further purification.

[0141]N-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butyl-D-seryl)-cis-4-(benzoylthymin-1-yl)-D-prolinewas obtained as a write solid [87%, starting from 1.46 mmol ofFmoc-D-Ser(O^(t)Bu)-D-Pro(cis-4-BzT)-ODpm]. ¹H NMR revealed the presenceof impurities, however, the crude product was used for the next stepwithout further purification.

[0142] General Procedure for Synthesis ofN-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butylseryl)-cis-4-(benzoylthymin-1-yl)prolinePentafluorophenyl Ester

[0143] A mixture of the N-Fmoc-dipeptide, pentafluorophenol (1.1 eq) andDCCI (1.1 eq) in dichloromethane was stirred at room temperature for 2-3hr. The precipitated dicyclohexylurea was filtered off, the solventremoved under reduced pressure and the residue purified by columnchromatography (SiO₂, dichloromethane:acetone or EtOAc).

[0144]N-(N-Fluoren-9-ylmethoxycarbonyt-0-t-butyl-L-seryl)-cis-4-(benzoylthymin-1-yl)-D-prolinewas obtained as a white solid [50%, starting from 1.46 mmol ofFmoc-L-Ser(O^(t)Bu)-D-Pro(cis-4-BzT)-ODpm] δ_(H) (200 MHz; CDCl₃) 1.15(9H, s, ^(t)Bu), 1.97 (3H, s, thymine CH₃), 2.30-2.44 and 2.88-3.04 [2H,2xm, CH₂(3′)], 3.45-3.55 (1H, m) 3.59-3.69 (1H, m) 3.96-4.05 (1H, m)[CH₂(5′) and Ser CH _(a)H_(b)], 4.15-4.27, 4.354.39, 4.43-4.56, 4.194.54(5H, m, unresolved Fmoc aliphatic CH, CH₂ and Ser CH_(a).H _(b)),4.67-4.88 [2H, m, CH(2′) and Ser C_(a)H], 5.28-5.35 [CH(4′)], 5.53-5.57(1H d J=8.2 Hz, peptide NH), 7.3 0-7.5 8 [m, CH(6), phenyl, Fmoc andbenzoyl aromatic CH], 7.63-7.70 (2H, m, Bz o-CH), 7.74-7.78 and7.90-7.94 (2×2H, 2xd, Fmoc aromatic CH); m/z (APCI+) 876 (M+H⁺, 100),819 (71), 771, 715, 690, 470, 310, 179, 122.

[0145]N-(N-Fluoren-9-ylmethoxycarbonyl-0-t-butyl-D-seryl)-cis-4-(benzoylthymin-1-yl)-D-prolinewas obtained as a white solid [20%, starting from 1.46 mmol ofFmoc-D-Ser(O^(t)Bu)-D-Pro(cis-4-BzT)-ODpm] δ_(H) (200 MMz; CDCl₃) 1.17(9H, s, ^(t)Bu), 1.97 (3H, s, thymine CH₃), 2.26-2.45 and 2.90-3.05 [2H,2xm. CH₂(3′)], 3.49-3.70(2H, m), 4.054.39 (m) [CH₂(5′), Fmoc aliphaticCH, CH₂ and Ser CH₂), 4.61-4.72(1H, m Ser C_(a)H), 4.85-4.93 [1H, m,CH(2′)], 5.30-5.39 [CH(4′)], 5.58-5.62 (1H, d J=8.2 Hz, peptide NH),7.23 [1H, s, CH(6)], 7.31-7.60 (m, phenyl, Fmoc and benzoyl aromaticCH), 7.63-7,71 (2H, m, Bz o-CH), 7.75-7.79 and 7.91-7.95 (2×2H, 2xd,Fmoc aromatic CH)

[0146] Solid Phase Synthesis of Chiral Peptide-nucleic Acids ContainingSeryl-D-proline Backbone using Fmoc/OBu-fragment Coupling Strategy

[0147] Synthesis of cPNA containing D-seryl-D-proline backbone wascarried out on 5 mmol scales on Novasyn TGR resin [preloaded withFmoc-Lys(Boc)—OH 0.23 mmol/g; 10-25 mg, ca. 2.5-5 pmol] using thedipeptide pentafluorophenyl esters in the presence of HOBT in DMF (4 eqeach, 3 h, rt) as described previously. The coupling reaction wasmonitored by measurement of the amounts of dibenzofulvene-piperidineadduct released upon deprotection at 300 nm which generally indicated95-100% efficiency. After the addition of the final residue wascompleted, the N-terminal Fmoc group was removed by 20% piperidine inDMF and the cPNA was released from the resin by treatment withtrifluoroacetic acid containing 5% anisole (ca. 1 ml for a 5 μmolsynthesis) at room temperature for 4-6 h with occasional agitation.After the specified period of time, the cleavage solution was dilutedwith diethyl ether (ten times the volume) and kept at −20° C. overnight.The suspension was then centrifuged at 13,000 rpm for 5 min. Afterdecanting the supernatant, the crude cPNA was washed with ether and thecentrifugation-wash process repeated 4-5 times. Finally the crude cPNAwas air dried and dissolved in 10% aqueous acetonitrile containing 0.1%trifluoroacetic acid. The crude solution was filtered and analysed orpurified by reverse phase hplc. The sample elution was carried out usinga gradient of water-acetonitrile containing 0.1% trifluoroacetic acid.The identity of the products were proved by ESI-MS. The yield of thecPNAs as determined by measurement of OD260 were 28 and 16% for LD-ST 10and DD-ST 10 respectively.

[0148]¹H NMR Experiment

[0149]¹H NMR study of a mixture of DD-ST10 and dA₁₀ was performed on aBruker AMX 500 spectrometer (500 MHz). The ¹H NMR spectra of DD-ST10 anddA₁₀ were recorded separately (at a concentration of 0.53 mM for theDD-ST10 and 0.67 mM for the dA₁₀ in 10% D₂O in H₂O). Upon addition of 20mol % of dA₁₀ (as a concentrated aqueous solution) to a solution of 0.53mM of DD-ST10 in 10% D₂O-H₂O, an immediate precipitation occurred.

REFERENCES

[0150] 1. E Uhlmann and A. Peyman, Chem Rev., 1990, 90(4), 543.

[0151] 2. a) P. Garner and J. U. Yoo, Tetrahedron Lett., 1993, 34, 1275;b) I. Lewis, Tetrahedron Lett., 1993, 34, 5697; c) A. Lenzi, G.Reginato, M. Taddei and E. Trifilieff, Tetrahedron Lett., 1995, 36,1717.

[0152] 3. P. E. Nielsen, M. Egholm, R. H. Berg and 0. Buchardt, Science,1991, 254, 1497.

[0153] 4. O. Almarsson, T. C. Bruice, J. Kerr and R. N. Zuckermann,Proc. Natl. Acad. Sci. USA, 1993, 90, 7518.

[0154] 5. P K T Lin and D M Brown, Nucleic Acids Research, 1989, 17,10373-83.

[0155] 6. J. Kovacs in The Peptides Vol 2, E. Gross and J. Meienhofered., Academic Press, New York, 1980, pp. 486-536.

[0156] 7. G. B. Fields and R. L. Noble, Int. J. Peptide Protein Res.,1990, 35, 161.

[0157] 8. T. W. Green and P. G. M. Wuts, Protecting Groups in OrganicSynthesis, 2nd edn., John Wiley & Sons, New York, 1991, p.328.

[0158] 9. Z. Tozuka & T. Takaya, J. Antibiotics, 1983, 36, 142

[0159] 10. G. C. Stalakatos, A. Paganon and L. Zervas, J.Chem. Soc. C,1966,1

[0160] 11. L. Kisfaludy and I. Schon, Synthesis, 1983, 325.

[0161] 12. M. L. Peterson and R. Vince J. Med. Chem., 1991, 34,, 2787.

[0162] 13. M. T. Chenon, R. J. Pugmire, D. M. Grant, R. P. Panzica andL. B. Townsend, J. Am.Chem. Soc., 1975, 97, 4627.

[0163] 14. R. R. Chauvette, R. A. Pennington, C. W. Ryan, R. D. E.Cooper, L. Jose, I. G. Wright, E. M. Heyningen, G. W. Huffmann J. Org.Chem., 1971, 36,1259.

[0164] 15. D. M. Brown, A. Todd and S. Varadarajan, J. Chem. Soc., 1956,2384.

[0165] 16. T. F. Jenny, K. C. Schneider and S. A. Benner, Nucleosides &Nucleotides, 1992, 11, 1957.

[0166] 17. a) E. Atherton and R. C. Sheppard, Solid Phase PeptideSynthesis, A Practical Approach. IRL Press, Oxford, 1989; b) G. B.Fields and R. L. Noble, Int. J. Peptide Protein Res., 1990, 35, 161.

[0167] 18. a) M. Goodman and K. C. Steuben, J. Am. Chem. Soc., 1962, 84,1279; b) M. Bodanszky, Principles of Peptide Synthesis, Springer-Verlag,Heidwlberg, 1984, p. 174.

[0168] 19. P. S. Miller, M. P. Reddy, ,A, Murakami, K R Blake, S.-B. Linand C. H. Agris, Biochemistry, 1986, 25, 5092.

[0169] 20. E. P. Stirchak, J. E. Summerton and D. D. Weller, J. Org.Chem., 1987, 52, 4202.

[0170] 21. D. D. Perrin and W. L. F. Amarego, Purification of LaboratoryChemicals, 3rd ed., Pergamon Press, Oxford, 1988.

[0171] 22. J. B. Miller, J. Org. Chem., 1959, 24, 560.

[0172] 23. E. Kaiser, R. L. Colescctt, C. D. Bossinger and P. I. Cook,Anal. Biochem., 1970, 34, 595.

TABLE 1 Electrospray mass spectral data of T₁₀ chiral peptide nucleicacids. Experimental values are the averaged mass derived from variousprotonated species in the mass spectra. Chiral Peptide Nucleic AcidM_(r) (found) M_(r) (calcd.) H-[Gly-D-Pro(trans-4-T)]₁₀-Lys- 2927.53 ±1.54 2927.88 [M] NH₂ 2968.29 ± 0.40 2965.97 [M−H+K] 3005.07 ± 0.533004.26 [M−2H+2K] H-[Gly-L-Pro(cis-4-T)]₁₀-Lys- 2929.02 ± 0.34 2927.88[M] NH₂ 2967.27 ± 0.97 2965.97 [M−H+K] 3005.88 3004.26 [M−2H+2K]H-[Gly-D-Pro(cis-4-T)]₁₀-Lys- 2929.88 ± 2.81 2927.88 [M] NH₂ 2965.52 ±0.36 2965.97 [M−H+K] 3004.38 ± 1.39 3004.26 [M−2H+2K] 3043.83 ± 0.663042.15 [M−3H+3K]

[0173] TABLE 2 Melting Temperatures Melting temperatures T_(m) (° C.)and % hypochromicity obtained from melting curves (recorded at 260 nm)of the hybrids of the cPNAs with poly(rA), poly(dA) and dA₁₀ in 150 mMsodium chloride, 10 mM sodium phosphate pH 7.0. The T_(m)S weredetermined from the maxima of the first derivative plots of thenormalised OD₂₆₀ against temperature. The T_(m) for the T₁₀oligodeoxyribonucleotide bound to poly(dA) is 27° C. at the same pH andionic strength. T_(m) with poly (rA) T_(m) with poly (dA) (° C.) and %(° C.) and % T_(m) with cPNA hypochromicity hypochromicity dA₁₀ (° C.)H-[Gly-D-Pro(cis- 72 (45%) 70 (28%) 61 4-T)]₁₀-LysNH₂ H-[Gly-L-Pro(cis-73 (36%) 69 (40%) 42 4-T)]₁₀-LysNH₂ H-[Gly-D-Pro(trans- No melting Nomelting No melting 4-T)]₁₀-LysNH₂ observed observed observed

1. A compound of formula (I):

where n is 1 or 2-200, B is a protected or unprotected base capable ofWatson-Crick or of Hoogsteen pairing, R is H, C₁-C₁₂ alkyl, C₆-C₁₂aralkyl or C₆-C₁₂ heteroaryl which may carry one or more substituentspreferably selected from hydroxyl, carboxyl, amine, amide, thiol,thioether or phenol. X is OH or OR′ where R′ is a protecting group or anactivating group or a lipophilic group or an amino acid or amino amideor nucleoside, Y is H or a protecting group or a lipophilic group or anamino acyl group or nucleoside.
 2. A compound as claimed in claim 1,wherein the structure is (II) or (Ill) where n, B, R, X and Y are asdefined in claim
 1.


3. A compound as claimed in claim 1 or claim 2, wherein B is a naturallyoccurring nucleobase selected from adenine, cytosine, guanine, thymineand uracil.
 4. A compound as claimed in any one of claims 1 to 3,wherein —CO—CHR—NH— is a residue of a naturally occurring amino acid. 5.A compound as claimed in any one of claims 1 to 4, wherein R is CH₂OH or(CH₂)₄NH₂ or H.
 6. A compound as claimed in claim 1, wherein n is 1, Bis a naturally occurring nucleobase selected from adenine, cytosine,guanine, thymine and uracil, R is H or CH₂OH or (CH₂)₄NH_(2.) X is OH orOR′, R′ is an activating group for example pentafluorophenyl, Y is H ora protecting group for example Fmoc.
 7. A compound as claimed in any oneof claims 1 to 5, wherein n is 2-200 preferably 5-30.
 8. A hybridcomprising two strands of which a first strand is a compound accordingto claim 7 and a second strand is an oligo- or poly-nucleotide ornucleic acid.
 9. A hybrid as claimed in claim 8, wherein the two strandsare hybridised to one another in a 1:1 molar ratio by base-specificWatson-Crick base pairing.
 10. A method of making the peptide nucleotideanalogue of formula (I), comprising the steps of: a) reacting anN-protected C-protected 4-hydroxy proline with a base selected fromN₃-protected thymine, N₆-protected adenine, N₄-protected cytosine,N₂—O₆-protected guanine and N₃-protected uracil. b) deprotecting theproline amino group of the product of a), c) reacting the product of b)with an N-protected amino acid, d) optionally removing protecting groupsfrom the product of c).
 11. A method as claimed in claim 10, wherein ina) 4-hydroxyproline in the form of a N-Boc/Dpm ester is reacted withN₃-benzoyl thymine, N,-benzoyl adenine, N₄-benzoyl cytosine,N₂-benzoyl-O₆-(4′-nitrophenylethyl)guanine or N₃-benzoyl uracil, and inc) an Fmoc amino acid ester is used.
 12. A method as claimed in claim 10or claim 11, wherein an N-protected C-protected trans-4-hydroxy prolineis used in a).
 13. A method of converting a peptide nucleotide analogueof formula (I) in which n is 1 into a peptide oligonucleotide of formula(l) in which n is 2-200, comprising the steps of: i) providing a supportcarrying primary amine groups, ii) coupling an N-protected peptidenucleotide analogue of formula (I) to the support, iii) removing theN-protecting group, iv) coupling an N-protected nucleotide analogue offormula (I) to the thus-derivatised support, v) repeating steps iii) andiv) one or more times, and vi) optionally removing the resulting peptideoligonucleotide from the support.
 14. A method as claimed in claim 13,wherein a pentafluorophenyl ester of the peptide nucleotide analogue isused in step ii) and iii).
 15. A compound of formula (IV)

where R² is H or a protecting group, R³ is H or a protecting groupcompatible with R², and B is a protected or unprotected heterocyclicbase.
 16. A compound as claimed in claim 15, wherein R² isdiphenylmethyl and R³ is t-butoxycarbonyl.
 17. A compound as claimed inclaim 15 or claim 16, wherein B is a protected or unprotected nucleobaseselected from adenine, cytosine, guanine, thymine and uracil.
 18. Acompound of formula (V)

where R² is diphenylmethyl, and R³ is t-butoxycarbonyl.
 19. A compoundas claimed in any one of claims 1 to 7, wherein at least one of B, R, Xand Y includes a signal moiety.